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

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

Our initial experiments involving the transgenic L. major lines overexpressing spike protein, either the full length S gene sequence or secreted S1/S2 subunits, showed that in each case the clones were attenuated in mice. New transgenic lines were generated: 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 Fn strain 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. Full-length spike protein was detected at expected band size (143 kDa) on blots for all clones of L. major transfectants but not in the wild-type (WT) culture samples. Ten thousand metacyclic promastigotes purified from WT L. major or Spike-L. major cultures were inoculated intradermally into both C57BL/6J mice ears. The full length Spike-L. major line and the S1/S2 secreting lines again failed to induce any visible ear lesions while the WT control produced ulcerated lesions. The attenuated recombinant parasites were again 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, although the frequencies were relatively low (spike L. major - 3.7% of CD4+ T cells were IFNg+ vs 0.52% for wt L. major). A PE-labeled peptide MHC Class 1 tetramer containing the dominant H-2K(b) spike epitope was used for detection of spike-specific CD8+ T cells in the infected mice. The frequencies of tetramer positive CD8+ T cells were not increased in either the ears or ear draining lymph nodes of mice infected with Spike-L. major compared to Wt L. major. Thus, mice infected with the transgenic parasites fail to mount a strong spike specific T cell response.