Novel SARS-CoV-2 vaccine candidates: Live, recombinant Leishmania

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Principal Investigator: David  Sacks
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2022
Award: $57,995
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. 
An L. major clone with the highest expression level of the spike protein (Spike-L. major) was selected  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.   Unexpectedly, the Spike-L. major cells failed to  induce any visible ear lesions while the WT control  produced ulcerated lesions.  L. major parasites over-expressing the secreted S1/S2 subunits were also  found to be fully attenuated in mice.  The recombinant parasites that were fully attenuated with respect to lesion development were nonetheless present in the ear at numbers that were not significantly reduced compared to the wild type strain or to a control recombinant strain expressing ovalbumin.     The lymph nodes from the LmSpike-infected mice contained a higher frequency of spike-specific IFNg+ CD4+ T cells compared to the WT-infected mice.

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