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Principal Investigator: David Sacks
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $22,137
Funding agency: National Institute of Allergy and Infectious Diseases
In order to overexpress SARS CoV-2 spike protein (S) in Leishmania, the full length S gene sequence was amplified by PCR from plasmid pCAGGS containing the Wuhan-Hu-1 spike and inserted into plasmid pSSU-NEO using a Gibson Assembly reaction, with the addition of an N-terminal myc tag and a C-terminal HA tag to evaluate spike cleavage into two subunits (S1 and S2) by potential Leishmania proteases. The generated plasmid pSSU-fullS was then linearized with PmeI and PacI enzymes and the purified construct was integrated into Leishmania major Friedlin strain and Leishmania tarentolae TarII strain 18S rRNA locus by transfection (4D-Nucleofector, Lonza). Positive transfectants were selected by geneticin (G418) antibiotic resistance.
Expression of SARS CoV-2 spike was confirmed by Western Blotting using anti-myc and anti-HA antibodies in the three clones tested for each species. Full-length spike protein was detected at expected band size (143 kDa) on blots for all clones of L. major and L. tarentolae transfectants but not in the wild-type (WT) culture samples. A larger protein band was also detected in all transfectants samples, which may be evidence of a glycosylated form of the spike protein, as this is a highly glycosylated protein in infected human cells. In addition, there is no evidence so far that there is specific cleavage between the S1 and S2 domains of the spike protein in Leishmania.
Next, an L. major clone with the highest expression level of the spike protein (Spike-L. major) was selected for a trial experiment to evaluate mouse immune response to the spike-overexpressing cells. Ten thousand metacyclics purified from WT L. major or Spike-L. major cultures were inoculated intradermally into both C57BL/6J mice ears using five animals in each group. Three weeks post-inoculation, the Spike-L. major cells have, unexpectedly, not yet induced any visible ear lesions while the WT control already produced ulcerated lesions. Further data points from the ongoing infection and future inoculation experiments may provide some clarification on this phenotype. At the endpoint of this experiment, the ears, draining lymph nodes, spleen, blood and bronchoalveolar lavage will be evaluated for evidence of anti-spike response.
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