Name: Jensen, Anja Home Country: Denmark Research Country: Project Period: 1998-2001   Title Identification of novel Leishmania antigens and development of a DNA vaccine against lesimaniasis   Abstract This thesis is based on work done from 1997 to August 2000 at Institute for Medical Microbiology and Immunology (Department of Parasitology), University of Copenhagen. The purpose of the work was to identify Leishmania antigens for subsequent use in Leishmaniasis vaccines. Leishmaniasis is not a single entity but comprises a wide range of human diseases. The clinical spectrum ranges from asymptomatic infections to infections, which carry a high mortality. The solid immunity observed following convalescence to cutaneous and visceral leishmaniasis has suggested that vaccination may prove a way of controlling Leishmania infections at a population level. Unfortunately, no vaccine is currently available. The "Tropical Disease Research programme" (WHO) is currently co-ordinating the development of second-generation leishmaniasis vaccines based on various different recombinant Leishmania proteins. These recombinant proteins have been produced in a number of laboratories around the world and are being tested in experimental murine Leishmania models in our laboratory and the laboratory of Dr. Manoel Baral Netto, Universitat Federal da Bahia, Brazil. An ancient practice of inoculating live Leishmania from the pus of an active cutaneous lesion to produce a self healing lesion in an inconspicuous site has been used as a form of vaccinating people. However, this kind of vaccination using live virulent parasites can be used only with Leishmania species that produce benign self-healing lesions. Since mid 1990 more than 1000 publications on DNA vaccines have appeared. The increasing number of studies using experimental animal models have demonstrated that plasmid DNA immunisation can promote effective immune responses against numerous viruses as well as parasitic infections such as malaria and leishmaniasis. To create a DNA vaccine it is essential to have a number of vaccine candidates. A number of different methods have been used previously in the attempt to isolate and select relevant Leishmania antigens for vaccine development some of which are T -cell blotting and traditional biochemical separation techniques used on Leishmania promastigotes. The strategy employed in this thesis was based upon screening of a cDNA library using plasma from donors infected or previously infected with Leishmania donovani. A total number of 40 clones were selected by use of this screening method and DNA sequenced. Of the 40 clones 35 showed similarity to the L. chagasi kinesin-like protein, two clones showed similarity to L. major Expressed Sequence Tags (EST) sequences and two clones did not show similarity to any published sequences. One clone was found to encode the Glucose Regulated Protein (GRP78), the endoplasmatic member of the 70-kDa heat shock protein family. The 1.14 kb cDNA clone of grp78 encodes a protein fragment of 380 amino acids with a predicted molecular mass of 42- kDa. Protein sequence analysis revealed 72-91 % homology with members of the 70-kDa heat shock protein family. The identity was being highest with the GRP78 of Trypanosoma cruzi. The L. major grp78 DNA sequence was PCR amplified using L. donovani specific primers and found to be 97.6% similar at the amino acid level. Like the Trypanosoma cruzi GRP78 both Leishmania GRP78 proteins contain a carboxy-terminal endoplasmic retention signal sequence (MDDL). Thegrp78 gene was found to be located on chromosome 15 in L. donovani,  L. major, and L. mexicana by pulse-field gel electrophoresis. Immunofluorescence on fixed and permeabilised promastigotes using GRP78 anti-serum showed a uniform staining in promastigotes with staining localised to reticular material throughout the cytoplasm and to perinuclear regions, suggesting that the protein is localised in the endoplasmic reticulum. Serological evaluation using recombinant GRP78 protein and plasma samples obtained in the Sudan revealed that 89% of individuals with VL, 78% with post kala-azar dermal leishmaniasis (PKDL), and 85% with CL had antibody reactivity to this novel Leishmania antigen. Plasma from healthy Sudanese donors living in an area endemic for malaria but free of leishmaniasis and plasma from healthy Danes were negative in the assay. Anti-GRP78 antibody were only infrequently found in Sudanese and Ghanaian malaria patients. In lymphoproliferative assays, peripheral blood mononuclear cells (PBMC) isolates from two of three donors previous infected with L. donovani and two of three donors previously infected with L. major showed a response to rGRP78, whereas Dine PBMC isolates from Danes did not proliferate. The grp78 DNA sequence was sub-cloned into an eukaryotic expression vector (pcDNA3.l). A resistant strain of mice, C57BL/6 mice were vaccinated with the naked DNA construct (pcDNAgrp78) or dendritic cells transfected with the construct and challenged with L. major parasites. Mice immunised with the naked DNA construct were clearly better at controlling the infection compared to the other groups included in the experiment and at week 7 all of the mice receiving the naked DNA construct had healed their lesions. By contrast, mice immunised with transfected dendritic cells or control groups of mice did not heal the lesions until week 9 and 10. Recombinant GRP78 protein was used to vaccinate BALB/c mice, which are highly susceptible to Leishmania and thus very difficult to protect. GRP78-GST was injected either alone or together with the Leishmania Parasite Surface Antigen-2 (PSA-2) protein, which is a leading vaccine candidate. The injection of GRP78-GST without adjuvant conferred partial protection at a level comparable to the protection achieved when using the recombinant protein with heat-killed Corynebacterium parvum An almost complete protection was achieved when GRP78-GST and PSA-2 were injected simultaneously. This immunisation scheme was just as effective in protecting BALB/c mice against L. major infection as was the standard vaccination protocol ofPSA-2 together with C. parvum. In contrast to the PSA-2 plus C. parvum injected mice and control groups of mice, which did not heat their lesions before the experiment was terminated, all BALB/c mice receiving GRP78-GST and PSA-2 had healed their lesions at week 9. Involved research institution(s) Centre for Medical Parasitology, University of Copenhagen Universidade Federal da Bahia, Brazil   Supervisor(s) Thor Theander, Centre for Medical Parasitology   Correspondence