Design and validation of VLP/mRNA-based vaccine strategies against SARS-CoV2

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Principal Investigator: paolo  lusso
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $67,461
Funding agency: National Institute of Allergy and Infectious Diseases

The project has yielded the following results:
1.	Synthesis of tail-modified SARS-CoV2 envelope spike (S) mRNAs to facilitate VLP formation with lentivirus core proteins. In order to facilitate the assembly of VLPs containing lentivirus (HIV-1 or SIV) core proteins, we have designed S proteins modified in their cytoplasmic tails (CT) by replacement of the natural tail with either the HIV-1 or SIV gp41 CT.  A truncated form of the lentivirus tails has been used, based on our original design for the HIV-1 vaccine, to promote a more efficient surface membrane expression.  Moderna Inc. has produced the specific mRNAs according to our design.
2.	Recombinant expression of the SARS-CoV2 envelope spike (S) glycoprotein in mammalian cells. The mRNA expressing the various CoV2 envelope spike (S) glycoproteins (native, HIV-CT and SIV-CT chimeric) produced by Moderna Inc. were tested in mammalian cells to verify their expression both in terms of efficiency and antigenic profile using commercially available monoclonal antibodies (mAbs) against the S protein.
3.	Production of VLPs. The spike protein mRNAs were co-transfected into mammalian cells in all possible combinations with mRNAs encoding HIV-1 or SIV Gag or Gag-Pol. The extracellular VLPs were harvested and concentrated by ultracentrifugation on sucrose cushions.
4.	Extensive characterization of VLPs. The produced VLPs have been extensively characterized both quantitatively and qualitatively using a wide array of physical and immunological assays, including virion capture by mAbs followed by ELISA for lentivirus core antigen quantification, Western blot and others.

The preliminary results indicate that VLPs comprising the CoV2 Spike protein and lentiretrovirus core can be efficiently produced and display the native-like form of the S-protein on their surface. Extensive characterization is still under way and will be followed by immunogenicity studies in small animal models.

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