Elsevier

Vaccine

Volume 23, Issue 9, 19 January 2005, Pages 1170-1177
Vaccine

Development of a recombinant Leishmania major strain sensitive to ganciclovir and 5-fluorocytosine for use as a live vaccine challenge in clinical trials

https://doi.org/10.1016/j.vaccine.2004.08.032Get rights and content

Abstract

To provide a safer live challenge strain for use in clinical vaccine trials, a double drug sensitive strain of Leishmania major was derived using advances in gene targeting technology by stably introducing into the chromosome a modified HSV-1 thymidine kinase gene (tk), conferring increased sensitivity to ganciclovir (GCV), and a Saccharomyces cerevisiae cytosine deaminase gene (cd), conferring sensitivity to 5-fluorocytosine (5-FC). In vitro studies showed that the homozygous L. major (tk–cd+/+) promastigotes were killed by either drug alone, and together the drugs acted synergistically. In vivo infection studies showed that progressively growing lesions in BALB/c mice, caused by L. major (tk–cd+/+), were completely cured by 2 weeks of treatment with either drug alone or in combination. Treated animals showed no signs of reoccurrence of infection for at least 4 months when the experiments were terminated.

Introduction

Leishmania is a protozoan parasite that causes a wide spectrum of clinical disease in humans. Leishmaniasis presents as either a disease of localized cutaneous lesions, that generally self-heal and lead to subsequent immunity, or as a chronic disseminating disease in the liver and spleen that is often fatal unless treated by chemotherapy. Leishmaniasis is endemic in 88 countries on five continents, Africa, Asia, Europe, North America and South America, with a total of 350 million people at risk. The total number of infected individuals has been estimated to be over 12 million with an estimated 1.5–2 million new cases per year [1]. Over the past several years, there has been a dramatic increase in reported cases of leishmaniasis. Epidemics of visceral leishmaniasis with a high rate of mortality continue to erupt, such as in the Sudan in the 1990s, where there were over 100,000 deaths with the majority (65%) in children [2]. Since 2002 there has been an ongoing epidemic of cutaneous leishmaniasis in Kabul, Afghanistan, with an estimated 200,000 cases currently reported [3]. In addition, Leishmania and HIV coinfection is an emerging disease in at least 30 countries worldwide including southern Europe where it has been reported that AIDS increases the risk of visceral leishmaniasis by 100–1000 times in endemic areas [4]. In 2002, the serious world health issues surrounding leishmaniasis were recognized by the World Health Organization (WHO) by the placement of leishmaniasis into the top category for research priorities of tropical diseases, with an emphasis on drug discovery and vaccine development [5].

Historically, the knowledge that immunity to leishmaniasis often develops upon resolution of disease in humans, led to early attempts at vaccine development, particularly against cutaneous leishmaniasis, using “leishmanization”, where live virulent parasites were inoculated. Leishmanization has been used as an immunization method for the prevention of cutaneous infection by Leishmania major since ancient times [6], [7], [8]. However, today this practice has been halted due to the subsequent development of a number of chronic cases that required medical treatment [8], [9]. In spite of these drawbacks, leishmanization is still currently being used as a protective measure in regions of Uzbekistan where leishmaniasis is highly endemic [10].

Over the past three decades a variety of approaches have been tried to develop a safer protective vaccine. The development of killed or irradiated promastigotes, recombinant antigens, recombinant live vectors and DNA, some of which have been reported to be total or partially protective in experimental animals and sub-human primates, has been recently reviewed [11]. Further testing of these vaccine candidates in the field is often carried out using randomized double blind field trials in highly endemic areas, that rely on natural infection as a challenge. Several thousand volunteers are required to gain statistically significant numbers. There have been several large field vaccine trials assessing the potential of heat killed promastigote preparations in combination with suitable adjuvants [12], [13], [14]. However, to date, the results from these studies have not identified an effective vaccine against leishmaniasis.

An alternate approach for large scale field testing of candidate vaccines and one that has proven successful in the development of vaccines for prevention of malaria [15] or viral diseases [16], [17] relies on a live challenge to insure consistency of infection rates and to reduce the number of participants in such vaccine studies. Organisms used for these live challenge studies were chosen to be highly sensitive to approved drug therapy in cases where the live challenge resulted in disease. Leishmanization has the potential to be used as a live challenge in Leishmania vaccine trials however, as discussed above, challenge with live organisms can lead to chronic disease with the added possibility of patients developing resistance to currently available drug therapies [8], [9]. The lack of a safe strain of Leishmania for use as a live challenge in such studies has been one of the critical limiting factors in the successful development of an effective vaccine for the prevention of leishmaniasis.

Gene targeting methods have been developed for Leishmania to either introduce foreign genes into the genome or to derive deficient strains (knock-out mutants) to assess the function of individual genes or gene families [18], [19], [20], [21]. The objective of this study was to develop a safer live challenge strain for use in clinical vaccine trials by using advances in gene targeting technology to stably introduce in the genome of L. major two drug sensitivity genes: a modified herpes simplex virus (HSV) thymidine kinase gene (tk), conferring increased sensitivity to ganciclovir (GCV) and acyclovir, and a Saccharomyces cerevisiae (S. cerevisiae) cytosine deaminase gene (cd), conferring sensitivity to 5-fluorocytosine (5-FC). In vitro and in vivo studies showed that the recombinant double drug sensitive L. major (tk–cd+/+) was sensitive to either drug alone and, when added together, the drugs acted synergistically.

Section snippets

Parasites

L. major strain MHOM/IR/76/ER [12], [13] was used in these studies and promastigotes (wild type) were maintained in liquid medium 199 supplemented with 10% defined heat inactivated fetal calf serum (Hyclone, Logan, USA). Cell growth assays for recombinant and wild type L. major were performed in media containing nourseothricin at a final concentration of 80 μg/mL (Werner Bio Agents, Jenacospeda, Germany) and hygromycin (hyg) at a final concentration of 60 μg/mL (Sigma–Aldrich, Oakville, Canada).

Generation of the tk–cd constructs for gene targeting

Isolation of the CD gene and construction of the TK–CD gene targeting fragment

The S. cerevisiae cd gene was chosen for use in Leishmania as previous studies reported that the yeast cd enzymatic activity was thermally stable at physiological temperatures [24]. A PCR approach was used to isolate the S. cerevisiae cd gene and the resulting 0.5 kb fragment was subcloned into the pGEMT Easy plasmid vector (Promega) and the complete DNA sequence was determined and was identical to the published sequence of FCY1 (NCBI ID:856175) predicting a protein sequence of 158 amino acids.

Discussion

A recombinant double drug sensitive strain, L. major (tk–cd+/+), was derived by integration of a genetically engineered HSV tk gene, conferring increased sensitivity to GCV and acyclovir, and the S. cerevisiae cd gene, conferring sensitivity to 5-fluorocytosine, into the genome of L. major for potential use as a live vaccine challenge in humans. The intergenic region of the L. major α-tubulin gene was cloned downstream of the tk and cd gene to provide constitutive expression of these genes

Acknowledgements

We are grateful to Margaret Black for generously providing the modified HSV tk75 gene. This study was supported by grants from the UNDP/World Bank/WHO Special Programme for Research & Training in Tropical Diseases (WRM) and the Canadian Institutes for Health Research grant number MOP 7399 (WRM).

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