Cloning, high-level gene expression and bioinformatics analysis of SP15 and LeIF from Leishmania major and Iranian Phlebotomus papatasi saliva as single and novel fusion proteins: a potential vaccine candidate against leishmaniasis

2020 ◽  
Author(s):  
Ali Bordbar ◽  
Massoud Amanlou ◽  
Kamran Pooshang Bagheri ◽  
Paul Donald Ready ◽  
Sahar Ebrahimi ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Protective Efficacy ◽  
Single Amino Acid ◽  
High Yield ◽  
Phlebotomus Papatasi ◽  
Vaccine Strategy ◽  
Bioinformatics Analyses ◽  
Gene Optimization ◽  
Immunogenic Proteins ◽  
Leishmania Parasites

Abstract Background Early exacerbation of cutaneous leishmaniasis is mainly affected by both the salivary and Leishmania parasite components. Little is known of the vaccine combination made by immunogenic proteins of sandfly saliva (SP15) with Leishmania parasites (LeIF) as a single prophylactic vaccine, namely SaLeish. Also, there are no data available to determine the species-specific sequence of SP15 isolated from the Iranian Phlebotomus papatasi. Methods Integrated bioinformatics and genetic engineering methods were employed to design, optimize and obtain a vector–parasite-based vaccine formulation in a whole-length fusion form of LeIF-SP15 against leishmaniasis. Holistic gene optimization was initially performed to obtain a high yield of pure ‘whole-SaLeish’ expression using bioinformatics analyses. Genomic and salivary gland RNAs of wild-caught P. papatasi were extracted and their complementary DNA was amplified and cloned into pJET vector. Results The new chimeric protein of whole-SaLeish and randomly selected transcripts of native PpIRSP15 (GenBank accession nos. MT025054 and MN938854, MN938855 and MN938856) were successfully expressed, purified and validated by immunoblotting assay. Furthermore, despite the single amino acid polymorphisms of PpIRSP15 found at positions Y23 and E73 within the population of wild Iranian sandflies, antigenicity and conservancy of PpIRSP15 epitopes remained constant to activate T cells. Conclusions The SaLeish vaccine strategy takes advantage of a plethora of vector–parasite immunogenic proteins with potential protective efficacy to stimulate both the innate and specific cellular immune responses against Leishmania parasites.

scholarly journals Vaccination with DNA Encoding the Immunodominant LACK Parasite Antigen Confers Protective Immunity to Mice Infected with Leishmania major

1997 ◽  
Vol 186 (7) ◽  
pp. 1137-1147 ◽  
Author(s):  
Sanjay Gurunathan ◽  
David L. Sacks ◽  
Daniel R. Brown ◽  
Steven L. Reiner ◽  
Hughes Charest ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Protective Immunity ◽  
Leishmania Major ◽  
Protective Efficacy ◽  
Dna Vaccination ◽  
Attractive Alternative ◽  
Dna Immunization ◽  
Dna Encoding ◽  
Ifn Γ

To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-γ (IFN-γ) production. Moreover, both the enhancement of IFN-γ production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8+ T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8+ T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.

A novel role for the peritrophic matrix in protecting Leishmania from the hydrolytic activities of the sand fly midgut

Parasitology ◽  
1997 ◽  
Vol 115 (4) ◽  
pp. 359-369 ◽  
Author(s):  
P. F. P. PIMENTA ◽  
G. B. MODI ◽  
S. T. PEREIRA ◽  
M. SHAHABUDDIN ◽  
D. L. SACKS
Keyword(s):  
Digestive Enzymes ◽  
Leishmania Major ◽  
Specific Inhibitor ◽  
Sand Fly ◽  
Peritrophic Matrix ◽  
Phlebotomus Papatasi ◽  
Hydrolytic Activities ◽  
Natural Vector ◽  
Parasite Survival

The role of the peritrophic matrix (PM) in the development of Leishmania major infections in a natural vector, Phlebotomus papatasi, was investigated by addition of exogenous chitinase to the bloodmeal, which completely blocked PM formation. Surprisingly, the absence of the PM was associated with the loss of midgut infections. The chitinase was not directly toxic to the parasite, nor were midgut infections lost due to premature expulsion of the bloodmeal. Most parasites were killed in chitinase-treated flies within the first 4 h after feeding. Substantial early killing was also observed in control flies, suggesting that the lack of PM exacerbates lethal conditions which normally exist in the blood-fed midgut. Early parasite mortality was reversed by soybean trypsin inhibitor. Allosamadin, a specific inhibitor of chitinase, led to a thickening of the PM, and also prevented the early parasite mortality seen in infected flies. Susceptibility to gut proteases was extremely high in transitional-stage parasites, while amastigotes and fully transformed promastigotes were relatively resistant. A novel role for the PM in promoting parasite survival is suggested, in which the PM creates a barrier to the rapid diffusion of digestive enzymes, and limits the exposure of parasites to these enzymes during the time when they are especially vulnerable to proteolytic damage.

scholarly journals Phlebotomus papatasi Antimicrobial Peptides in Larvae and Females and a Gut-Specific Defensin Upregulated by Leishmania major Infection

2021 ◽  
Vol 9 (11) ◽  
pp. 2307
Author(s):  
Barbora Kykalová ◽  
Lucie Tichá ◽  
Petr Volf ◽  
Erich Loza Telleria
Keyword(s):  
Gene Expression ◽  
Antimicrobial Peptides ◽  
Leishmania Major ◽  
Larval Instar ◽  
Gut Bacteria ◽  
Sand Fly ◽  
Control Group ◽  
Phlebotomus Papatasi ◽  
Major Infection ◽  
Defensin Gene

Phlebotomus papatasi is the vector of Leishmania major, causing cutaneous leishmaniasis in the Old World. We investigated whether P. papatasi immunity genes were expressed toward L. major, commensal gut microbes, or a combination of both. We focused on sand fly transcription factors dorsal and relish and antimicrobial peptides (AMPs) attacin and defensin and assessed their relative gene expression by qPCR. Sand fly larvae were fed food with different bacterial loads. Relish and AMPs gene expressions were higher in L3 and early L4 larval instars, while bacteria 16S rRNA increased in late L4 larval instar, all fed rich-microbe food compared to the control group fed autoclaved food. Sand fly females were treated with an antibiotic cocktail to deplete gut bacteria and were experimentally infected by Leishmania. Compared to non-infected females, dorsal and defensin were upregulated at early and late infection stages, respectively. An earlier increase of defensin was observed in infected females when bacteria recolonized the gut after the removal of antibiotics. Interestingly, this defensin gene expression occurred specifically in midguts but not in other tissues of females and larvae. A gut-specific defensin gene upregulated by L. major infection, in combination with gut-bacteria, is a promising molecular target for parasite control strategies.

scholarly journals Generation of a CRISPR/Cas9-Based Vector Specific for Gene Manipulation in Leishmania major

2019 ◽  
Author(s):  
Ghodratollah SALEHI SANGANI ◽  
Vahid JAJARMI ◽  
Ali KHAMESIPOUR ◽  
Mahmoud MAHMOUDI ◽  
Abdolmajid FATA ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Leishmania Major ◽  
Gene Knockout ◽  
Ease Of Use ◽  
Selection Marker ◽  
Gene Manipulation ◽  
Resistance Proteins ◽  
U6 Promoter ◽  
Overlap Extension ◽  
Leishmania Parasites

Background: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. Results: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

Immunogenicity and protective efficacy of orally administered recombinant Lactococcus lactis expressing surface-bound HIV Env

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 223-228 ◽  
Author(s):  
Ke-Qin Xin ◽  
Yuka Hoshino ◽  
Yoshihiko Toda ◽  
Shizunobu Igimi ◽  
Yoshitsugu Kojima ◽  
...  
Keyword(s):  
Immune Responses ◽  
Lactococcus Lactis ◽  
Potential Role ◽  
Protective Efficacy ◽  
Oral Immunization ◽  
Vaccine Strategy ◽  
Regional Lymph Nodes ◽  
Viral Loads ◽  
Recombinant Lactococcus Lactis ◽  
Further Development

Abstract This study investigates whether genetically modified orally administered Lactococcus lactis (L lactis) could be used as an HIV vaccine. L lactis is immunogenic and extremely safe when delivered orally. We created a recombinant L lactis vector expressing the envelope protein of HIV on its cell surface. Oral immunization with this vector induced high levels of HIV-specific serum IgG and fecal IgA antibodies. Cell-mediated immune responses also were generated in both the regional lymph nodes and the spleen. Dendritic cells are readily infected by L lactis and appear to play a potential role in mediating the development of these immune responses. The protective efficacy of this vaccine strategy was demonstrated by challenging mice intraperitoneally with an HIV Env–expressing vaccinia virus. Their viral loads were 350-fold lower than those of control mice. These findings support the further development of L lactis–based HIV vaccines. (Blood. 2003; 102:223-228)

Occurrence of Leishmania major in sandfly urine

Parasitology ◽  
1999 ◽  
Vol 118 (5) ◽  
pp. 455-460 ◽  
Author(s):  
J. SÁDLOVÁ ◽  
P. VOLF
Keyword(s):  
Leishmania Major ◽  
Fluorescence Assay ◽  
Blood Agar ◽  
Phlebotomus Papatasi ◽  
Free Swimming ◽  
Specific Fluorescence ◽  
Low Intensity

Promastigotes of Leishmania major were frequently detected in the urine droplets discharged by infected Phlebotomus papatasi and P. duboscqi females during feeding. Parasites were present in the urine of 37·5% P. papatasi and 16·1% P. duboscqi females, even in those with low intensity gut infections. Free-swimming forms (elongated nectomonads, short slender promastigotes and metacyclic forms) predominated in excreted droplets. Viability of excreted parasites was proved by cultivation on blood agar, and the presence of metacyclic forms in urine droplets was confirmed by specific fluorescence assay with 3F12 antibodies. While the release of promatigotes from the anus of the sandfly was frequent, these were rarely egested from the mouth-parts of sandfly females (1·3% for P. duboscqi and 0% for P. papatasi) fed on microcapillaries, even if the females were heavily infected. The possible role and significance of the discharge of parasites in sandfly urine are discussed.

LB Broth-lyophilized Rabbit Anti-sheep Cell Haemolysin as a Simple Culture Medium for Cultivation of Leishmania Major Promastigotes

2019 ◽  
Author(s):  
Vahid Nasiri ◽  
Farnoosh Jameie ◽  
Habibollah Paykari
Keyword(s):  
Blood Cell ◽  
Culture Medium ◽  
Leishmania Major ◽  
Low Cost ◽  
Protozoan Parasites ◽  
Causative Agents ◽  
Genus Leishmania ◽  
Leishmania Parasites ◽  
Growth Ability

Background and Aims: The protozoan parasites of the genus Leishmania are the causative agents of various clinical diseases. Different methods of cultivation of Leishmania parasites are available. In the present study, the efficacy of the LB broth with rabbit lyophilized anti-sheep red blood cell haemolysin was evaluated in the cultivation of promastigotes of Leishmania major. Materials and Methods: Conventional LB broth medium was prepared and autoclaved for 15 min at 121°C and then lyophilized rabbit anti-sheep cell haemolysin was added at 1-10% final concentrations. The efficacy of the medium was evaluated by assessing the growth ability and replication patterns of the promastigotes of Leishmania major. Results: Medium with 1-10% lyophilized rabbit haemolysin supported the growth of the parasites and can be used for cultivation of Leishmania parasites with acceptable In vivo infectivity for research purpose. Conclusions: The ability of the parasites to survive and proliferate in the presence of lyophilized rabbit haemolysin indicates that this material is a good nutritional source. This study opens a new way to make low-cost medium that can be used in cultivation of Leishmania parasites

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