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A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing. ​

Literature Information ​

DOI10.1038/s41467-020-17154-z
PMID32651371
JournalNature communications
Impact Factor15.7
JCR QuartileQ1
Publication Year2020
Times Cited52
KeywordsLeishmaniasis, vaccine, CRISPR gene editing, Leishmania major, immune response
Literature TypeJournal Article, Research Support, N.I.H., Intramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.
ISSN2041-1723
Pages3461
Issue11(1)
AuthorsWen-Wei Zhang, Subir Karmakar, Sreenivas Gannavaram, Ranadhir Dey, Patrick Lypaczewski, Nevien Ismail, Abid Siddiqui, Vahan Simonyan, Fabiano Oliveira, Iliano V Coutinho-Abreu, Thiago DeSouza-Vieira, Claudio Meneses, James Oristian, Tiago D Serafim, Abu Musa, Risa Nakamura, Noushin Saljoughian, Greta Volpedo, Monika Satoskar, Sanika Satoskar, Pradeep K Dagur, J Philip McCoy, Shaden Kamhawi, Jesus G Valenzuela, Shinjiro Hamano, Abhay R Satoskar, Greg Matlashewski, Hira L Nakhasi

TL;DR ​

This study introduces a CRISPR-edited Leishmania major strain (LmCen-/-) as a second-generation vaccine for leishmaniasis, addressing the safety concerns associated with traditional leishmanization by being lesion-free and antibiotic-resistant marker-free. Mice immunized with LmCen-/- showed no lesions and robust protection against infection, suggesting its potential for safe advancement to human vaccine trials and effective immunization against this neglected tropical disease.

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Leishmaniasis · vaccine · CRISPR gene editing · Leishmania major · immune response

Abstract ​

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

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Primary Questions Addressed ​

  1. What are the potential long-term effects of using the LmCen-/- strain in human vaccine trials?
  2. How does the immune response generated by the LmCen-/- strain compare to traditional leishmanization methods?
  3. What challenges might arise in the regulatory approval process for a CRISPR-edited vaccine like LmCen-/-?
  4. In what ways could the development of a markerless vaccine influence future vaccine strategies for other diseases?
  5. What are the implications of using antibiotic resistance in vaccine strains for public health and safety?

Key Findings ​

Research Background and Purpose ​

Leishmaniasis, a neglected tropical disease caused by Leishmania protozoa, affects millions globally, with no effective vaccines currently available. Traditional vaccination methods, such as leishmanization using live L. major, are not practiced due to safety concerns, including the development of skin lesions. This study aims to develop a safer second-generation leishmanization vaccine using a CRISPR-engineered, marker-free L. major strain (LmCen−/−) that induces immunity without causing disease.

Main Methods/Materials/Experimental Design ​

The researchers employed CRISPR-Cas9 gene editing to create the LmCen−/− strain, which lacks the centrin gene, crucial for parasite proliferation. The process involved:

  1. Designing guide RNAs targeting the centrin gene.
  2. Transfecting these into L. major promastigotes using a CRISPR vector.
  3. Selecting for clones with the centrin gene deletion based on reduced growth rates.
  4. Confirming the deletion and absence of off-target effects through whole-genome sequencing.

The experimental design included immunizing mice with LmCen−/− and subsequently challenging them with wild-type L. major through both needle and sand fly bites to assess protective immunity.

Mermaid diagram

Key Results and Findings ​

  1. Centrin Deletion Confirmation: Whole-genome sequencing confirmed the precise deletion of the centrin gene with no off-target mutations.
  2. Immunogenicity and Safety: Mice immunized with LmCen−/− did not develop lesions and showed a robust immune response, comparable to traditional leishmanization.
  3. Protection Against Challenge: Immunized mice were protected from both needle and sand fly challenges, with significantly reduced lesion sizes and parasite burdens compared to non-immunized controls.

Main Conclusions/Significance/Innovation ​

The study successfully demonstrates that the CRISPR-engineered LmCen−/− strain is a safe and effective candidate for a live attenuated vaccine against cutaneous leishmaniasis. This approach overcomes the safety issues associated with traditional leishmanization, potentially paving the way for human clinical trials and offering a new strategy for combating this neglected disease.

Research Limitations and Future Directions ​

  • Limitations: The study primarily used murine models, which may not fully replicate human responses. Long-term effects and potential immune responses in humans remain to be evaluated.
  • Future Directions: Further studies should focus on advancing LmCen−/− to clinical trials, investigating its efficacy in diverse populations, and understanding the immune mechanisms involved in protection. Additionally, exploring the use of other virulence genes for further attenuation could enhance vaccine safety and efficacy.

References ​

  1. Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG. - Matthew E Rogers;Thomas Ilg;Andrei V Nikolaev;Michael A J Ferguson;Paul A Bates - Nature (2004)
  2. KSAC, a defined Leishmania antigen, plus adjuvant protects against the virulence of L. major transmitted by its natural vector Phlebotomus duboscqi. - Regis Gomes;Clarissa Teixeira;Fabiano Oliveira;Phillip G Lawyer;Dia-Eldin Elnaiem;Claudio Meneses;Yasuyuki Goto;Ajay Bhatia;Randall F Howard;Steven G Reed;Jesus G Valenzuela;Shaden Kamhawi - PLoS neglected tropical diseases (2012)
  3. Vaccination using live attenuated Leishmania donovani centrin deleted parasites induces protection in dogs against Leishmania infantum. - Jacqueline Araújo Fiuza;Sreenivas Gannavaram;Helton da Costa Santiago;Angamuthu Selvapandiyan;Daniel Menezes Souza;Lívia Silva Araújo Passos;Ludmila Zanandreis de Mendonça;Denise da Silveira Lemos-Giunchetti;Natasha Delaqua Ricci;Daniella Castanheira Bartholomeu;Rodolfo Cordeiro Giunchetti;Lilian Lacerda Bueno;Rodrigo Correa-Oliveira;Hira L Nakhasi;Ricardo Toshio Fujiwara - Vaccine (2015)
  4. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. - Nathan C Peters;Jackson G Egen;Nagila Secundino;Alain Debrabant;Nicola Kimblin;Shaden Kamhawi;Phillip Lawyer;Michael P Fay;Ronald N Germain;David Sacks - Science (New York, N.Y.) (2008)
  5. Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites. - Colby Zaph;Jude Uzonna;Stephen M Beverley;Phillip Scott - Nature medicine (2004)
  6. Research priorities for elimination of visceral leishmaniasis. - Greg Matlashewski;Byron Arana;Axel Kroeger;Ahmed Be-Nazir;Dinesh Mondal;Shan Golam Nabi;Megha Raj Banjara;Murari Lal Das;Baburam Marasini;Pradeep Das;Graham Medley;Abhay Satoskar;Hira Nakhasi;Daniel Argaw;John Reeder;Piero Olliaro - The Lancet. Global health (2014)
  7. Leishmaniasis worldwide and global estimates of its incidence. - Jorge Alvar;Iván D Vélez;Caryn Bern;Mercé Herrero;Philippe Desjeux;Jorge Cano;Jean Jannin;Margriet den Boer; - PloS one (2012)
  8. Centrin gene disruption impairs stage-specific basal body duplication and cell cycle progression in Leishmania. - Angamuthu Selvapandiyan;Alain Debrabant;Robert Duncan;Jacqueline Muller;Poonam Salotra;Gannavaram Sreenivas;Jeffrey L Salisbury;Hira L Nakhasi - The Journal of biological chemistry (2004)
  9. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. - Phillip Scott;Fernanda O Novais - Nature reviews. Immunology (2016)
  10. BEDTools: a flexible suite of utilities for comparing genomic features. - Aaron R Quinlan;Ira M Hall - Bioinformatics (Oxford, England) (2010)

Literatures Citing This Work ​

  1. Human Vaccines & Immunotherapeutics: news. - Human vaccines & immunotherapeutics (2020)
  2. Characterization of a new Leishmania major strain for use in a controlled human infection model. - Helen Ashwin;Jovana Sadlova;Barbora Vojtkova;Tomas Becvar;Patrick Lypaczewski;Eli Schwartz;Elizabeth Greensted;Katrien Van Bocxlaer;Marion Pasin;Kai S Lipinski;Vivak Parkash;Greg Matlashewski;Alison M Layton;Charles J Lacey;Charles L Jaffe;Petr Volf;Paul M Kaye - Nature communications (2021)
  3. Protein methyltransferase 7 deficiency in Leishmania major increases neutrophil associated pathology in murine model. - Juliana Alcoforado Diniz;Mariana M Chaves;Slavica Vaselek;Rubens D Miserani Magalhães;Rafael Ricci-Azevedo;Renan V H de Carvalho;Lucas B Lorenzon;Tiago R Ferreira;Dario Zamboni;Pegine B Walrad;Petr Volf;David L Sacks;Angela K Cruz - PLoS neglected tropical diseases (2021)
  4. Inoculation of the Leishmania infantum HSP70-II Null Mutant Induces Long-Term Protection against L. amazonensis Infection in BALB/c Mice. - Manuel Soto;Laura Ramírez;José Carlos Solana;Emma C L Cook;Elena Hernández-García;José María Requena;Salvador Iborra - Microorganisms (2021)
  5. Revival of Leishmanization and Leishmanin. - Thalia Pacheco-Fernandez;Greta Volpedo;Sreenivas Gannavaram;Parna Bhattacharya;Ranadhir Dey;Abhay Satoskar;Greg Matlashewski;Hira L Nakhasi - Frontiers in cellular and infection microbiology (2021)
  6. Nano- and Microformulations to Advance Therapies for Visceral Leishmaniasis. - Devika M Varma;Elizabeth A Redding;Eric M Bachelder;Kristy M Ainslie - ACS biomaterials science & engineering (2021)
  7. A review of the leishmanin skin test: A neglected test for a neglected disease. - Jessica Carstens-Kass;Kayla Paulini;Patrick Lypaczewski;Greg Matlashewski - PLoS neglected tropical diseases (2021)
  8. Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis. - Subir Karmakar;Nevien Ismail;Fabiano Oliveira;James Oristian;Wen Wei Zhang;Swarnendu Kaviraj;Kamaleshwar P Singh;Abhishek Mondal;Sushmita Das;Krishna Pandey;Parna Bhattacharya;Greta Volpedo;Sreenivas Gannavaram;Monika Satoskar;Sanika Satoskar;Rajiv M Sastry;Timur Oljuskin;Telly Sepahpour;Claudio Meneses;Shinjiro Hamano;Pradeep Das;Greg Matlashewski;Sanjay Singh;Shaden Kamhawi;Ranadhir Dey;Jesus G Valenzuela;Abhay Satoskar;Hira L Nakhasi - Communications biology (2021)
  9. Protective CD4+ Th1 cell-mediated immunity is reliant upon execution of effector function prior to the establishment of the pathogen niche. - Leah S Hohman;Zhirong Mou;Matheus B Carneiro;Gabriel Ferland;Rachel M Kratofil;Paul Kubes;Jude E Uzonna;Nathan C Peters - PLoS pathogens (2021)
  10. Human leishmaniasis vaccines: Use cases, target population and potential global demand. - Stefano Malvolti;Melissa Malhame;Carsten F Mantel;Epke A Le Rutte;Paul M Kaye - PLoS neglected tropical diseases (2021)

... (42 more literatures)

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