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

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

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. At the endpoint of this experiment at 12 wks p.i., fewer than 2,000 Spike-L. major cells were present in the ear, while the resolving lesions in the ears of mice infected with w.t. L. major contained more than 50,000 parasites. A minimal T cell response against Spike protein was detected in the draining LN of ears infected with Spike-L. major.