Biomarkers of Attenuation in the Leishmania donovani Centrin Gene Deleted Cell Line—Requirements for Safety in a Live Vaccine Candidate



Robert Duncan*, a, Ranadhir Deyaa, Keiko Tomiokaaa, b, Heather Hairstona, c, Angamuthu Selvapandiyana, d, Hira L. Nakhasia
a Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA
b Hepatitis Viruses Section, LID, NIAID, Rockville, MD 20852, USA
c Infectious Disease Clinical Research Program, National Naval Medical Center, Bethesda, MD 20889, USA
d Institute of Molecular Medicine, 254 Okhla Industrial Estate, Phase III, New Delhi,11002a, India


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© 2009 Duncanet al;

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, 1401 Rockville Pk. HFM310, MD 20892, USA; Tel: 301-496-1651; Fax: 1-480-7928; E-mail: Robert.Duncan@fda.hhs.gov


Abstract

Biomarkers of the attenuated phenotype are needed to develop live attenuated parasites into safe Leishmania vaccines. The centrin-1 gene deleted strain of Leishmania donovani (LdCEN1-/- ) shows promise as a vaccine candidate. To identify genes whose expression patterns are indicators of attenuation, the LdCEN1-/- line was compared to wild type by gene expression microarray. Two genes, one coding for a 27kDa protein (p27) and another coding for putative Argininosuccinate Synthase (AS) have such expression patterns. Both genes express a higher RNA level in the amastigote stage than in the promastigote stage of wild type cells; however they are down-regulated in the LdCEN1-/- amastigote cells. Western blots indicated that the AS protein level is also reduced in the LdCEN1-/- amastigotes, while the p27 protein level is not reduced even when its mRNA level has diminished. Northern and Western blot analysis with these two biomarkers showed that LdCEN1 parasites recovered after five weeks of infection in mice had the same expression pattern as they had prior to infection and episomal expression of centrin in the LdCEN1-/- cells restored normal expression of both genes. Measurement of the expression of these two genes in infected macrophages by RT-PCR indicated the same pattern as in cultured cells. Therefore, both the mRNA and/or the protein levels of these two genes could be used as biomarkers of attenuation to monitor the safety of the LdCEN1-/- cell line as it is developed as a potential vaccine.

Keywords: Leishmaniasis vaccines, Parasites, Vaccines, Live attenuated, Gene expression, Attenuated phentype biomarkers.