Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis.
ABSTRACT The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmania donovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund's adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1×10(7) L. donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.
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ABSTRACT: Studies were carried out on the mechanisms by which B lymphocytes are polyclonally activated to secrete antibodies during visceral leishmaniasis. Crude extracts of Leishmania donovani, the aetiological agent of this disease, of Leishmania mexicana amazonensis, the etiological agent of cutaneous leishmaniasis, and of Herpetomonas muscarum, a related non-pathogenic organism, all contain components which cause strong in vitro polyclonal activation of hamster spleen cells leading to the production of antibodies. However, in vivo, only hamsters infected with L. donovani develop hypergammaglobulinaemia due to B cell polyclonal activation. Hamsters injected with the crude extracts of leishmania or infected with L. mexicana amazonensis do not manifest these alterations in their B cell response. Furthermore spleen cells of hamsters infected with L. donovani became unresponsive to stimulation with the T cell mitogen phytohaemagglutinin (PHA) by day 10 of infection, whereas their response to concanavalin A (Con A) was preserved. The decreased lymphocyte response to PHA coincided with the augmentation of the PFC/spleen ratio. In contrast, spleen cells from hamsters infected with L. mexicana amazonensis, responded normally to both mitogens throughout the course of infection. These results suggest that the hypergammaglobulinaemia present in visceral leishmaniasis may be the consequence of an inbalance of regulatory T cells, possibly associated with a direct stimulation of hamster B cells by L. donovani components.Clinical & Experimental Immunology 03/1985; 59(2):427-34. · 3.41 Impact Factor
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ABSTRACT: Patients with visceral leishmaniasis produce high levels of immunoglobulin, but the specificities of antibodies produced are not well characterized. In an effort to identify leishmania antigens that are specific to Leishmania species or are cross-reactive with other parasitic protozoa, we have cloned and characterized full-length genomic and cDNA clones encoding a Leishmania chagasi acidic ribosomal antigen, LcP0, recognized during human infections. The protein is homologous to the Trypanosoma cruzi and human ribosomal proteins TcP0 and HuP0, respectively. Unlike most higher eukaryotes, but similar to TcP0, LcP0 has a C-terminal heptapeptide sequence resembling those of the archaebacterial acidic (P-like) proteins. The highly charged C-terminal acidic domain of LcP0 contains a serine residue typically found in most eukaryotes but lacking in all T. cruzi P proteins we have characterized thus far. L. chagasi-infected individuals as well as those with T. cruzi infections have antibodies cross-reactive with recombinant LcP0 and TcP0 as well as HuP0. However, the properties of anti-P0 antibodies in T. cruzi and L. chagasi infection sera are quite different. Through the use of synthetic peptides, we showed that while T. cruzi infection anti-TcP0 antibodies are exclusively directed against the C-terminal domain of TcP0, L. chagasi infection sera contain antibodies reactive with epitopes other than the C-terminal sequence of LcP0. Thus, anti-LcP0 antibodies in L. chagasi infection sera represent the first characterized deviation from the restricted immunodominant C-terminal epitope involved in the generation of anti-P0 antibodies following infection or autoimmune diseases.Infection and Immunity 06/1994; 62(5):1643-51. · 4.07 Impact Factor
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ABSTRACT: In order to identify a specific recombinant antigen of Leishmania donovani with potential use for diagnosis, a cDNA library was constructed in lambda ZAP II expression vector. On screening the cDNA library using pooled sera from Indian patients with kala azar, 20 antibody reactive clones were identified. These were subcloned into pBluescript phagemid by an in vivo excision procedure. The molecular weights of the expressed recombinant proteins varied from 15 to 70 kDa and the cDNA insert sizes varied from 0.5 kb to the largest size of approximately 2.0 kb which was designated as the E2b clone. The nucleotide sequencing revealed that 50% of the clones had sequence homology to the heat shock protein gene of L. donovani. The serological studies conducted with the kala azar positive sera and sera from healthy laboratory workers using the recombinant protein from the E2b clone and having sequence homology to Ldhsp 70, indicated that although all the kala azar sera was positive, 12 of 20 healthy individuals also showed antibodies against the recombinant hsp70, indicating that this antigen is not suitable for serological diagnosis of kala azar.Immunology and Cell Biology 11/1995; 73(5):446-51. · 3.93 Impact Factor