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Contents lists available at ScienceDirect

Vaccine

j ournal homepage: www.elsevier.com/locate/vaccine

1 Advancing a vaccine to prevent disease and

a,b,c,d,e,f,∗ a,b,d a,b,d

2 Q1 Peter J. Hotez , Coreen M. Beaumier , Portia M. Gillespie ,

a,b,d f a,b,c,d,e,f

3 Ulrich Strych , Tara Hayward , Maria Elena Bottazzi

a

4 National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA

b

5 Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

c

6 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA

d

7 and Texas Children’s Hospital Center for Vaccine Development, Houston, TX, USA

e

8 Department of Biology, Baylor University, Waco, TX, USA

f

9 Sabin Vaccine Institute, Washington, DC, USA

10

a r a

2111 t i c l e i n f o b s t r a c t

12

13 Article history: A human hookworm vaccine is under development and in clinical trials in Africa and the Americas. The

14 Available online xxx

vaccine is comprised of two recombinant proteins encoding Na-GST-1 and Na-APR-1, respectively, formu-

15 lated on alum. It elicits neutralizing antibodies that interfere with establishment of the adult hookworm

16 Keywords:

in the gut and the ability of the parasite to feed on . The vaccine target product profile is focused on

17 Hookworm

the of children to prevent hookworm and anemia caused by .

18 Vaccine

It is intended for use in low- and middle-income countries where hookworm is highly endemic and

19 Necator

responsible for at least three million disability-adjusted life years. So far, the human hookworm vaccine

20 Neglected tropical disease

is being developed in the non-profit sector through the Sabin Vaccine Institute Product Development

Partnership (PDP), in collaboration with the HOOKVAC consortium of European and African partners.

Ultimately, the vaccine will be incorporated into health systems as part of an elimination strategy for

and other neglected tropical diseases, and as a means to reduce global poverty and

address Sustainable Development Goals.

© 2016 World Health Organization. Published by Elsevier Ltd. This is an open access article under the

CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

22Q3 Human hookworm infection is a neglected tropical disease infection occurs when the larval stages penetrate the skin of a 39

23 caused predominantly by the parasite Necator ameri- human host. The primary adverse effect of infection, anemia, dis- 40

24 canus [1]. Recent estimates indicate that approximately 439 million proportionately occurs in children and pregnant women with lower 41

25 people are infected with hookworm worldwide, with the majority iron reserves compared to other populations [5,6]. Hookworm is 42

26 of cases found in the developing regions of South Asia (140 million hyperendemic among some pediatric populations in sub-Saharan 43

27 cases), Sub-Saharan Africa (118 million), Southeast Asia (77 mil- Africa where in countries such as Sierra Leone or Togo one-third 44

28 lion), East Asia (64.5 million), and the Latin American and Caribbean of the population under the age of 20 is infected [7]. Children with 45

29 region (30 million) [2,3]. In these areas, hookworm disease is a chronic hookworm infection experience anemia and cognitive and 46

30 major cause of iron-deficiency anemia, a consequence of the adult developmental delays with resultant reductions in school perfor- 47

31 hookworm’s ability to extract blood from the intestinal mucosa and mance, attendance and future wage earnings [8,9]. Approximately 48

32 submucosa [4]. 7 million pregnant women in sub-Saharan Africa – almost one third 49

33 The Global Burden of Disease Study 2010 (GBD 2010) esti- of annual pregnancies in Africa – are also infected, making hook- 50

34 mated that hookworm is responsible for a loss of 3.2 million worm disease one of the most common complications of pregnancy 51

35 disability adjusted life years (DALYs), making it one of the leading in that part of the world [5]. Moreover, many of these individuals – 52

36 neglected tropical diseases (along with schistosomiasis and leish- both children and pregnant women – are co-infected with malaria, 53

37 maniasis) in terms of disease burden [2] and a leading cause of thereby exacerbating anemia and its sequelae [10]. 54

38 anemia in large parts of Africa and Oceania [3,4] (Fig. 1). Hookworm The primary approach to hookworm control is mass drug admin- 55

istration with a single annual tablet of either of the anthelminthics 56

(400 mg) or (500 mg). Single-dose 57

∗ mebendazole, however, has yielded low cure rates, particularly 58

Corresponding author at: National School of Tropical Medicine, Baylor College

with repeated use [11–13]. One comprehensive meta-analysis 59

Q2 of Medicine, Houston, TX, USA. Tel.: +1 7137981199.

showed no impact of mebendazole treatment on improving anemia 60

E-mail address: [email protected] (P.J. Hotez).

http://dx.doi.org/10.1016/j.vaccine.2016.03.078

0264-410X/© 2016 World Health Organization. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/

by-nc-nd/3.0/).

Please cite this article in press as: Hotez PJ, et al. Advancing a vaccine to prevent hookworm disease and anemia. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.078

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Fig. 1. Distribution of human hookworm infection.

(Reproduced with permission from [5], Brooker et al., PLoS NTD).

Table 1

Development status of current vaccine candidates (POC = proof of concept trial).

Candidate name/identifier Preclinical Phase I Phase II POC Phase III

Na-GST-1 X

Na-APR-1 X

Na-GST-1 and Na-APR-1 Co-administered X

61 in hookworm-affected communities [6]. Similarly for albenda- studies showed that sera from vaccinated dogs protected non- 94

62 zole, drug failure has also been reported, though less often [14]. vaccinated dogs from hookworm challenge [21]. However, clinical 95

63 Moreover, children can re-acquire hookworm several months after trials of hookworm recombinant larval have revealed this 96

64 treatment, especially in areas of high transmission [15,16]. These approach to be unfeasible because of a high prevalence of IgE anti- 97

65 observations may explain a recent finding from GBD 2013 study bodies to larval macromolecules such as Na-ASP-2 among some 98

66 that overall global hookworm prevalence has remained essentially hookworm endemic populations [22]. 99

67 unchanged over the last 20 years, while the prevalence of other Proteins, especially enzymes, required for adult hookworm 100

68 neglected tropical diseases such as lymphatic filariasis, ascariasis, blood-feeding may hold promise as an alternative strategy for 101

69 and trachoma has decreased by 25–33% over the same time period hookworm vaccine development [23,24] (Fig. 2). This approach 102

70 [17]. There is, therefore, a need for new technologies to achieve was used successfully for a veterinary vaccine against the blood- 103

71 better control of hookworm infection, particularly if the world is feeding trichostrongyle, Haemonchus contortus that infects sheep 104

72 to meet the proposed targets set by the London Declaration for and cattle [25]. The enzymes required for diges- 105

73 neglected tropical diseases and the follow-up World Health Assem- tion and heme detoxification in have been identified, 106

74 bly resolution 66.12 [18]. A safe and effective anti-hookworm cloned, expressed and shown to elicit protective antibodies [23,24]. 107

75 vaccine, as a complement to conventional chemotherapy, may Unlike the larval stage antigens there is no evidence that these 108

76 provide a cost-effective means of reaching this goal [19] (Table 1). enzymes induce IgE antibodies. In the case of N. americanus, sev- 109

eral enzymes have been identified and developed into recombinant 110

77 1. Biological feasibility for vaccine development immunogens, including aspartic protease-hemoglobinase, Na-APR- 111

1 (a critical enzyme for hemoglobin digestion) and glutathione 112

78 As hookworm infection does not typically confer immunity, S-transferase-1 (Na-GST-1) (a unique form of the enzyme used 113

79 there is so far no natural immunological correlate of protection on for parasite heme detoxification) [23,24]. Both have demonstrated 114

80 which to base a program for vaccine development. Hookworms are efficacy in immunization/challenge studies in dogs. In the case of 115

81 strong immunomodulators from the onset of infection, enabling Na-APR-1, the vaccine induced neutralizing antibodies against mul- 116

82 them to persist in their host for years. Even with chemotherapy, tiple heterologous strains of hookworm [26]. 117

83 reinfection is the rule. Furthermore, the prevalence and inten-

84 sity of infection increases with age, adding more evidence that 2. General approaches to vaccine development for low and 118

85 hookworm fails to elicit robust acquired immunity [20]. Biologi- middle income country markets 119

86 cal feasibility for vaccine development was first demonstrated by

87 the relative success of a commercial canine vaccine that consisted The selection strategy for a human hookworm vaccine 120

88 of a radiation-attenuated infective larval stage. The vaccine – mar- is based on several key criteria: (1) efficacy in animal trials, (2) 121

89 keted for dogs in the United States in the 1970s – achieved high absence of pre- antigen-specific IgE among endemic 122

90 levels of protection against infection with populations, (3) feasibility of scaled-up protein manufacturing 123

91 and consequent anemia, but was ultimately discontinued due to using low-cost expression systems such as yeast, bacteria or plants 124

92 the high cost of production and complex storage and distribution and (4) a plausible mechanism of protection [24]. From approx- 125

93 issues. Before its discontinuation, though, passive antibody transfer imately two-dozen proteins that are putatively involved in the 126

Please cite this article in press as: Hotez PJ, et al. Advancing a vaccine to prevent hookworm disease and anemia. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.078

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Fig. 2. Enzymes required for parasite blood feeding. Adult worms in the gut ingest blood, and parasite hemolysins drill pores into the erythrocytes, releasing hemoglobin into

the parasite gut lumen (step 1). Hemoglobin is digested by the hierarchical and ordered cascade of hemoglobinases (APR-1, an aspartic protease, CP-3, a cysteine protease, and

MEP-1, a metalloproteinase) lining the brush border membrane of the parasite gut (step 2). The globin peptides and free amino acids that are released following hemoglobin

digestion are absorbed into the gut cells, putatively being transported by OPT1 (step 3). Free heme is detoxified by the action of glutathione S-transferase (GST-1) (step 4).

Antibodies that could be induced by vaccination to neutralize the function of target proteins and interrupt blood feeding are shown.

(Reproduced with permission from [24] Hotez et al., Nature Reviews Microbiology).

127 hookworm blood-feeding process, the two previously mentioned through the US Food and Drug Administration (FDA). The latter two 161

128 antigens, Na-APR-1 and Na-GST-1, are the leading candidates for pathways are complicated by the finding that hookworm infec- 162

129 clinical development. The eventual goal is to license a vaccine tion is not endemic to the US or Europe except in small focal 163

130 that contains either or both of the recombinant forms of these areas. However, review by the EMA or FDA is recognized as high- 164

131 immunogens in formulation with an aluminum hydroxide adjuvant level assurance of product quality and safety, and could facilitate 165

®

132 (Alhydrogel ). Clinical trials are also evaluating whether an addi- approval by national regulatory authorities in hookworm disease- 166

133 tional adjuvant is required to achieve acceptable immunogenicity. endemic countries. Clinical endpoints for the human hookworm 167

134 These adjuvants are synthetic Toll-like receptor (TLR) agonists, such vaccine are being developed in parallel with parasitological end- 168

135 as, glucopyranosyl lipid A (GLA) and CpG oligodeoxynucleotide. points including, quantitative fecal egg counts (corresponding to 169

136 One of the cited reasons for the canine vaccine’s commercial fail- worm number), and fecal blood loss. Neutralizing anti-enzyme 170

137 ure was that it did not provide sterilizing immunity, even though antibodies are also being developed as potential surrogate corre- 171

138 this would not be a requirement for a human hookworm vaccine to lates of immunity. 172

139 deliver a clinical benefit. This is because the pathology of hookworm

140 corresponds mainly with intestinal blood loss, which is 4. Status of vaccine R&D activities 173

141 proportional to worm burden. The more important goal of a suc-

142 cessful vaccine, then, is reduce the burden of infection and thereby The preclinical downselection criteria that lead to the clinical 174

143 reduce blood and nutrient loss to a subclinical level. Thus, among development of Na-GST-1 and Na-APR-1 have been reviewed else- 175

144 the proposed features of the vaccine’s target product profile (TPP) is where [23,24]. In essence, these two immunogens were the most 176

145 an efficacy of at least 80% not in preventing infection entirely, but in efficacious in reducing hookworm burden and blood loss following 177

146 preventing moderate and heavy infections caused by N. americanus challenge of hamsters with N. americanus or dogs with A. caninum. 178

147 and the resulting intestinal blood loss and anemia. Additionally, the Both candidates are now in full-scale cGMP production and in phase 179

148 TPP outlines a vaccine intended for at risk children under the age 1 clinical trials. Na-GST-1 is being expressed in the yeast Pichia 180

◦ ◦

149 of 10, stable between 2 C and 8 C and administered at no more pastoris [33,34] while Na-APR-1 is being produced in the Nicotiana 181

150 than two doses by intramuscular injection concurrently with other tobacco plant [35]. Each molecule is undergoing separate phase 1 182

151 childhood [23]. testing as an alum formulation. Additionally, Na-GST-1, plus alum, 183

is being evaluated either with an aqueous formulation of the TLR 184

152 3. Technical and regulatory assessment 4 agonist, GLA, or the TLR-9 agonist, CpG deoxyoligonucleotide. 185

Na-APR-1 plus alum is being evaluated with GLA. 186

153 The monovalent Na-GST-1 and Na-APR-1 vaccines are currently In addition, to phase 1 clinical testing, the Sabin PDP is also 187

154 in phase 1 clinical trials in and the US [27–32]. A co- developing a hookworm larval challenge model in human volun- 188

155 administration trial with both vaccines is underway in Gabon [29]. teers vaccinated with one or both antigens [30]. Early results to 189

156 Three different strategies for regulatory approval have been pro- determine feasibility of developing the challenge model in human 190

157 posed: (1) registration in an endemic country where the vaccine volunteers will be available in 2016. The Sabin PDP has a parallel 191

158 will be manufactured at an industrial scale (e.g. Brazil) followed discovery program for new antigens screened from recently com- 192

159 by WHO prequalification, (2) use of the article 58 procedure of pleted hookworm genome project [36]. Additionally, a large part 193

160 the European Medicines Agency (EMA) or (3) regulatory approval of the human hookworm vaccine development efforts advancing 194

Please cite this article in press as: Hotez PJ, et al. Advancing a vaccine to prevent hookworm disease and anemia. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.078

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200 Universität Tübingen, CERMEL (Centre de Recherches Médicales

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Please cite this article in press as: Hotez PJ, et al. Advancing a vaccine to prevent hookworm disease and anemia. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.078