Approaches for the identification of potential excreted/secreted proteins ofLeishmania majorparasites

Leishmaniaparasites are able to survive in host macrophages despite the harsh phagolysosomal vacuoles conditions. This could reflect, in part, their capacity to secrete proteins that may play an essential role in the establishment of infection and serve as targets for cellular immune responses. To characterizeLeishmania majorproteins excreted/secreted early after promastigote entry into the host macrophage, we have generated antibodies against culture supernatants of stationary-phase promastigotes collected 6 h after incubation in conditions that partially reproduce those prevailing in the parasitophorous vacuole. The screening of anL. majorcDNA library with these antibodies led us to isolate 33 different cDNA clones that we report here. Sequence analysis revealed that the corresponding proteins could be classified in 3 groups: 9 proteins have been previously described as excreted/secreted inLeishmaniaand/or other species; 11 correspond to known proteins already characterized inLeishmaniaand/or other species although it is unknown whether they are excreted/secreted and 13 code for unknown proteins. Interestingly, the latter are transcribed as shown by RT-PCR and some of them are stage regulated. TheL. majorexcreted/secreted proteins may constitute putative virulence factors, vaccine candidates and/or new drug targets.

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Naloxone Diminishes the Virulence and Modifies the Cellular Immune Responses of BALB/c Mice Infected with Leishmania major

Acta Parasitologica ◽

10.1007/s11686-020-00308-w ◽

2020 ◽

Author(s):

Mohammad Hossein Alimohammadian ◽

Farhad Riazi-Rad ◽

Mahsa Asadi-Tat ◽

Sima Darabi ◽

Haiedeh Darabi ◽

...

Keyword(s):

Immune Responses ◽

Leishmania Major ◽

Cellular Immune Responses ◽

Cellular Immune

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Cellular immune responses induced in vitro by Ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides

Onderstepoort Journal of Veterinary Research ◽

10.4102/ojvr.v83i1.1170 ◽

2016 ◽

Vol 83 (1) ◽

Cited By ~ 5

Author(s):

Nontobeko Thema ◽

Alri Pretorius ◽

Selaelo I. Tshilwane ◽

Junita Liebenberg ◽

Helena Steyn ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Recombinant Proteins ◽

Mononuclear Cells ◽

Secreted Proteins ◽

Ehrlichia Ruminantium ◽

Cellular Immune Responses ◽

Ifn Γ ◽

Cellular Immune

Secreted proteins are reported to induce cell-mediated immunity characterised by the production of interferon-gamma (IFN)-γ. In this study three open reading frames (ORFs) (Erum8060, Erum7760, Erum5000) encoding secreted proteins were selected from the Ehrlichia ruminantium (Welgevonden) genome sequence using bioinformatics tools to determine whether they induce a cellular immune response in vitro with mononuclear cells from needle and tick infected animals. The whole recombinant protein of the three ORFs as well as four adjacent fragments of the Erum5000 protein (Erum5000A, Erum5000B, Erum5000C, Erum5000D) were successfully expressed in a bacterial expression system which was confirmed by immunoblots using anti-His antibodies and sheep sera. These recombinant proteins were assayed with immune sheep and cattle peripheral blood mononuclear cells (PBMCs), spleen and lymph node (LN) cells to determine whether they induce recall cellular immune responses in vitro. Significant proliferative responses and IFN-γ production were evident for all recombinant proteins, especially Erum5000A, in both ruminant species tested. Thus overlapping peptides spanning Erum5000A were synthesised and peptides that induce proliferation of memory CD4+ and CD8+ T cells and production of IFN-γ were identified. These results illustrate that a Th1 type immune response was elicited and these recombinant proteins and peptides may therefore be promising candidates for development of a heartwater vaccine.

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Characterization, cloning and immunogenicity of antigens released by lung-stage larvae of Schistosoma mansoni

Parasitology ◽

10.1017/s003118209900431x ◽

1999 ◽

Vol 118 (6) ◽

pp. 583-594 ◽

Cited By ~ 47

Author(s):

R. HARROP ◽

P. S. COULSON ◽

R. A. WILSON

Keyword(s):

Immune Responses ◽

Schistosoma Mansoni ◽

Protective Immunity ◽

Expression System ◽

Cdna Clones ◽

Cellular Immune Responses ◽

Potential Source ◽

Cellular Immune ◽

High Degree ◽

Expression Libraries

Lung-stage schistosomula are the target of protective immunity in mice vaccinated with attenuated cercariae of Schistosoma mansoni. Therefore, proteins present at this developmental stage, and in particular those which are secreted, are a potential source of novel vaccine candidates. However, little information is available about such molecules. Here we describe the cDNA clones identified by screening expression libraries with serum raised against proteins released by lung-stage schistosomula. In total, 11 different cDNA species were identified, 6 of which have been described previously in S. mansoni; these included fructose 1,6-bisphosphate aldolase and Sm21.7 which together accounted for two-thirds of all positive clones. Of the 5 newly described schistosome genes, 1 cDNA had a high degree of homology to the s5a subunit of 26S proteasomes, most significant being with the human protein. The remaining 4 clones showed no significant homologies to any genes sequenced previously. Fructose 1,6-bisphosphate aldolase, Sm21.7, the proteasome homologue and 1 unknown clone (A26) have been expressed in a bacterial expression system and serum produced against each recombinant protein. Immunolocalization showed fructose 1,6-bisphosphate aldolase, Sm21.7 and the proteasome homologue to be most abundant in muscle cells whilst clone A26 was distributed throughout many tissues, but was most abundant in the tegument. Analysis of the cellular immune responses of vaccinated mice showed 3 of the 4 expressed clones to be highly immunogenic, inducing the secretion of large quantities of the Th1-type cytokine interferon gamma.

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