DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica
Authors: Kambiz Kamyab-Hesari
Fabiano Oliveira
Jesus G Valenzuela
Amir Mizbani
Fatemeh Zali
Sima Habibzadeh
Yasaman Taslimi
Elham Gholami
Alireza Sadeghipour
Shaden Kamhawi
Safoora Gharibzadeh
Tahereh Taheri
Sima Rafati
Claudio Meneses
Negar Seyed
Nasim Gholami
Fatemeh Khadir
Daniel Omid Bakhadj
Mohammad Ali Mazlomi
Journal: PLoS Neglected Tropical Diseases (2019)
Discipline: IMMUNOLOGY
Keywords: leishmaniasis; sand flies; Phlebotomus; salivary proteins; PsSP9
Affiliations: Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran., Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran., Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America., Department of Epidemiology and Biostatistics, Pasteur Institute of Iran, Tehran, Iran., Research Centre for Emerging and Reemerging Infectious Disease, Pasteur Institute of Iran, Tehran, Iran., Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland., Department of Dermatopathology, Razi Hospital, Tehran University of Medical Sciences, Tehran, Iran., Department of Pathology, Hazrat-e-Rasoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran.

Abstract:  Background: The vector-borne disease leishmaniasis is transmitted to humans by infected female sand flies, which transmits Leishmania parasites together with saliva during blood feeding. In Iran, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) major and L. tropica, and their main vectors are Phlebotomus (Ph.) papatasi and Ph. sergenti, respectively. Previous studies have demonstrated that mice immunized with the salivary gland homogenate (SGH) of Ph. papatasi or subjected to bites from uninfected sand flies are protected against L. major infection. Methods and results: In this work we tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN-γ expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. Two months post infection, protection was associated with a significant increase in the ratio of IFN-γ to IL-5 expression in the dLN compared to controls. Conclusion: This study demonstrates that while immunity to the whole Ph. sergenti saliva does not induce a protective response against cutaneous leishmaniasis in BALB/c mice, PsSP9, a member of the PpSP15 family of Ph. sergenti salivary proteins, provides protection against L. tropica infection. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens. 

Retracted: No

PubPeer comments:
 Figure 1. * Blue boxes: Lanes 5 (VR1020-PsSP52 (32.5 KDa)) and 8 (VR1020-PsSP41 (37.4 KDa)) are more similar than expected if one of the two strips is mirrored. * Red boxes: Lanes 6 (VR1020-PsSP54 (18.4 KDa)) and 10 (VR1020-PsSP15 (18.4 KDa)) are more similar than expected * Teal, brown, purple, green ellipses: Unexpected repetitive patterns within the same blot strip. There might be a good explanation for this (e.g., a forceps indentation?), but hopefully the authors can reply with some insights. Image contrast enhanced to bring out details. ![file] , 
 CORRECTION ARTICLE Front. Immunol., 12 August 2019 | https: doi.org 10.3389 fimmu.2019.01828 There are errors in the Funding statement. The correct Funding statement is as follows: This work was funded by Pasteur Institute of Iran (Grant no. 752), Iran National Science Foundation (ID 940007) and International Center for Genetic Engineering and Biotechnology (ICGEB, Grant no. CRP IRN15-02) to Sima Rafati and also partially supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (JGV, SK, FO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation and interpretation of the manuscript. , 
 Dear Elisabeth M Bik This is in response to your query regarding Figure 1 of our manuscript u201cDNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropicau201d in PLoS Neglected Tropical Diseases. Question: Blue boxes: Lanes 5 (VR1020-PsSP52 (32.5 KDa)) and 8 (VR1020-PsSP41 (37.4 KDa)) are more similar than expected if one of the two strips is mirrored. Answer: Lane 5 and lane 8 represent different proteins with different molecular weights. Firstly, if the image was mirrored, we would also see the smear of Lane 5 on Lane 8 which we do not see demonstrating that the strips are different. Also, the molecular weight of the protein in Lane 5 is closer to the 31 kDa molecular weight marker while the protein in lane 8 is closer to the 45 molecular weight marker, showing that these two molecules are different and that they are not mirrored. Question: Red boxes: Lanes 6 (VR1020-PsSP54 (18.4 KDa)) and 10 (VR1020-PsSP15 (18.4 KDa)) are more similar than expected Answer: Proteins on lanes 6 and lane 10 are proteins of the same molecular weight that belong to the same family of proteins, and therefore they should produce bands of a similar (nearly identical) size in a gel or western blot. However, the background on Lane 6 and Lane 10 is clearly different demonstrating that they are not the same strips but different runs. Question: Teal, brown, purple, green ellipses: Unexpected repetitive patterns within the same blot strip. There might be a good explanation for this (e.g., a forceps indentation?), but hopefully the authors can reply with some insights. Answer: These marks are present on other strips as well and are likely specks of dust or possibly forceps marks. Actually, you can see the same type of indentation in Lane 12 and also many of these type of small indentations in Lanes 6, 9 and 10. However, these marks do not represent a protein in the figure or in our analysis. Kind regards Sima Rafati , 
 #3 Dr. Rafati: Thank you for responding. After reviewing the published figures, I believe there are good reasons for readers to share Dr. Biku2019s concerns. Could you please post the original uncropped scans? I did not see this in the supporting information. You may post a link here to an external site (such as Imgur.com or Google Drive) to provide readers access to larger original images. *** ![file] , 
 Dear Dr. Rafati, The more I look at some of these blot strips, the more unexpected repeats I appear to see. Here is a comparison of blots 6 and 10 in more detail, with the contrast a bit eenhanced. In particular blot 6 appears to show the same areas multiple times, as highlighted with boxes of the same color. ![file] To resolve the issue of the molecular weight, it would help if you could post the original blot strips, including the top and bottom edges, and put it the order with which they were run on the original gel. , 
 Here is an image forensics analysis by ImageTwin to support some of our findings: ![file] , 
 #4 ![file] ![file] ![file] ![file] , 
 Dear Dr. Rafati, Thank you for posting the original blots here, but not all of them appear to correspond to the published blots. Strips #3, 4, 5, 10, 13, and 14 look similar to the published photo, but the other lanes (1, 2, 6, 7, 8, 9, 11, and 12) appear to show different strips. Of note, the strips that appear to look duplicated (6 and 8) do not seem to correspond with the originals. So my concerns remain, unfortunately. Could you please check again? , 
 Dear Dr. Bik We have been appreciative of the posted comments requesting clarification for some figures in our paper and we think that they has been adequately addressed. We responded and provided evidence that our blots, though not perfect, were processed, run, and presented correctly. Thank you for your interest in our paper. ,