Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials

doi:10.1016/j.vaccine.2005.04.040

Vaccine

Volume 23, Issue 39, 15 September 2005, Pages 4754-4764

https://doi.org/10.1016/j.vaccine.2005.04.040 Get rights and content

Abstract

The ASP-2 protein secreted by infective larvae of the human hookworm, Necator americanus, is under development as a recombinant vaccine. Recombinant Na-ASP-2 was expressed in Pichia pastoris, and the purified protein was characterized. At the 60 L scale, the 21.3 kDa recombinant protein was produced at a yield of 0.4 g/L. When formulated with Alhydrogel^® and injected into rats to determine immunological potency, three 50 μg doses of the formulated recombinant protein elicited geometric mean antibody titers up to 1:234,881. Rat anti-Na-ASP-2 antibody recognized larval-derived ASP-2 and also inhibited larval migration through skin in vitro. The processes developed and tested for the high yield production of recombinant Na-ASP-2 provide a foundation for clinical vaccine development.

Introduction

Human hookworm infection caused by Necator americanus or Ancylostoma duodenale is a major cause of iron deficiency anemia and protein malnutrition in the developing world and a leading cause of parasitic disease burden [1], [2]. An estimated 740 million people are infected in areas of rural poverty in the tropics and subtropics resulting in up to 65,000 deaths and 22 million disability-adjusted life years [3], [4]. The major approach to hookworm control relies on reducing morbidity through the frequent and periodic use of benzimidazole anthelminthic (BZAs) drugs, which can temporarily remove adult parasites in the human gastrointestinal tract [4], [5]. Such treatment for school-aged children leads to improvements in health and educational achievement [4]. However, there are reasons why exclusive reliance on BZAs for controlling hookworm infection may not be effective. Among them, post-treatment hookworm re-infection rates are high in areas of high transmission [6], and the efficacy of BZAs can diminish with increasing and frequent use [7]. It has been further suggested that the emergence of BZA drug resistance is possible [7]. School-based programs are also not expected to interrupt hookworm transmission [8].

In order to develop new tools for the control of hookworm infection, studies were undertaken to develop recombinant hookworm vaccines. Based on previous success in the development of canine hookworm vaccines from third-stage infective larvae (L3) that were attenuated by X-irradiation [9], our antigen discovery efforts focused on the identification and isolation of critical L3-secreted molecules that mediate protective immunity [10]. These included a novel class of cysteine-rich secretory proteins known as the Ancylostoma secreted proteins or ASPs [10], [11], [12], [13], [14]. The ASPs belong to the pathogenesis-related (PR) protein superfamily whose members are found in a wide variety of animals, plants and fungi where they are typically produced in response to stress or injury, including invasion by pathogens [15]. Although the function of the hookworm larval ASPs is not known, the observation that they are released by L3 upon receiving host-specific stimuli suggests that these proteins are released during host entry and function in the transition from the environment to a parasitic existence [13].

ASP-2 was selected for evaluation as a recombinant vaccine candidate based on human immunoepidemiologic studies and laboratory animal vaccine trials [16], [17], [18], [19]. These data led to the selection of ASP-2 from the human hookworm N. americanus for further development. Here we describe the cloning of the Na-asp-2 gene and the scale-up expression of the purified recombinant Na-ASP-2 protein in the yeast, Pichia pastoris. We further describe the immunological potency of Na-ASP-2 and the cross-reactivity of antibody against the recombinant protein with the native, parasite-derived protein. The studies here also confirm the importance of Na-ASP-2 in hookworm tissue migration. These data lay the foundation for the clinical development of the Na-ASP-2 Hookworm Vaccine.

Section snippets

Molecular cloning of Na-asp-2 cDNA and transformation into Pichia pastoris

A cDNA library was constructed with mRNA from L3 of a Chinese strain of N. americanus [20], and probed with a heterologous cDNA fragment of Ac-asp-2 cloned from the dog hookworm Ancylostoma caninum [12]. The Ac-asp-2 pBluescript plasmid was cut with XhoI and BamHI, releasing a 739 bp fragment (28–767 bp). The fragment was gel purified (Qiagen) and randomly labeled with α-³²[P]-dCTP using a Rediprime labeling kit (Amersham). Approximately 5 × 10⁵ plaques of N. americanus cDNA library were screened

Identification and cloning of Na-asp-2

Two positive clones were obtained by screening the N. americanus L3 cDNA library with the radiolabeled Ac-asp-2 cDNA (28–767 bp) heterologous probe. Each clone contained a 731 bp cDNA encoding an open reading frame (ORF) of 206 amino acids with 65% amino acid identity with Ac-ASP-2. The Na-asp-2 cDNA sequence contained a 3′ poly(A) tail but no translation initiation codon (ATG) at the 5′-end. To identify additional Na-asp-2 cDNAs, further screening of 5 × 10⁵ plaques was undertaken using a probe

Discussion

Here we describe the expression, purification and characterization of Na-ASP-2, a lead candidate antigen under development as a human hookworm vaccine. We also show that anti-Na-ASP-2 antibody inhibits larval migration in vitro, providing evidence for the role of this protein in host tissue migration by N. americanus L3.

Phylogenetic analysis of the four hookworm ASP-2 molecules studied to date (one from N. americanus and three from different members of the genus Ancylostoma) reveals that they

Acknowledgements

We wish to thank Drs. Allan Saul and Louis Miller of the Malaria Vaccine Development Branch of the NIAID, NIH for allowing us to use their fermentation facilities. These studies were supported by the Human Hookworm Vaccine Initiative of the Bill and Melinda Gates Foundation and the Sabin Vaccine Institute. JB is supported by an International Research Scientist Development Award (1K01 TW00009) and AL is supported by an RD Wright Career Development Award from the National Health and Medical

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Between Nov 8 and Dec 14, 2018, 26 volunteers were screened, of whom 23 enrolled in the trial. The first immunisation was conducted on Dec 18, 2018. 23 volunteers were randomly assigned (15 to the intervention group and eight to the placebo group). Egg load after challenge was lower in the intervention group than the placebo group (geometric mean 571 eggs per g [range 372–992] vs 873 eggs per g [268–1484]); however, this difference was not statistically significant (p=0·10). Five volunteers in the intervention group developed a severe skin rash, which was associated with 40% reduction in egg counts after challenge (geometric mean 742 eggs per g [range 268–1484] vs 441 eggs per g [range 380–520] after challenge; p=0·0025) and associated with higher peak IgG1 titres.
To our knowledge, this is the first study to describe a protective effect of short-term exposure to hookworm larvae and show an association with skin response, eosinophilic response, and IgG1. These findings could inform future hookworm vaccine development.
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Citation Excerpt :
The CAP superfamily are widely distributed in eukaryotes including hookworm. Members of this superfamily in hookworms include Ancylostoma-secreted proteins (ASPs) previously isolated from A. caninum (Hawdon et al., 1996), A. ceylanicum (Goud et al., 2004), and N. americanus (Goud et al., 2005). Although members of this superfamily are widely spread in hookworm secretome (Morante et al., 2017), their functions are generally unknown, except for neutrophil inhibitory factor (NIF), which can block neutrophil adhesion to endothelial cells (Moyle et al., 1994), and Ac-HPI, which can inhibit platelets function (Del Valle et al., 2003).
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¹: Both GNG and MEB contributed equally to the manuscript.

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Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials

Abstract

Introduction

Section snippets

Molecular cloning of Na-asp-2 cDNA and transformation into Pichia pastoris

Identification and cloning of Na-asp-2

Discussion

Acknowledgements

Adv Parasitol

Trends Parasitol

Trans R Soc Trop Med Hyg

Trans R Soc Trop Med Hyg

Adv Parasitol

Int J Parasitol

J Biol Chem

Mol Biochem Parasitol

Curr Opin Genet Dev

Int J Parasitol

J Mol Biol

Vaccine

Int J Parasitol

Anal Biochem

J Biol Chem