Broad-spectrum RNA prophylactic and therapeutic interventions for SARS-CoV-2

Open Access
- Author:
- Gontu, Abhinay
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 04, 2024
- Committee Members:
- Anthony Schmitt, Professor in Charge/Director of Graduate Studies
Costas Maranas, Outside Unit Member
Marco Archetti, Outside Field Member
Bhushan Jayarao, Co-Chair & Major Field Member
Suresh Kuchipudi, Chair & Dissertation Advisor
Alberto Gino Lorenzoni Calvo, Outside Field Member - Keywords:
- RNA
SARS-CoV-2
Trans-amplifying mRNA vaccine
RNA mini-genome
SARS-CoV-2 variants - Abstract:
- Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease-2019 (COVID-19) pandemic is undergoing continuous evolution, leading to the emergence of mutants capable of evading prior immunity and undermining current vaccines and therapeutics. Hence, there is an urgent need to develop SARS-CoV-2 vaccines and therapeutics that are effective against the current and future variants. The goal of this thesis was to develop a broad-spectrum RNA-based SARS-CoV-2 vaccine and therapeutic. While mRNA vaccines have played a pivotal role in mitigating the impact of COVID-19, conventional mRNA vaccines face limitations in terms of cost and scalability due to their high antigenic mRNA load per dose, hindering widespread distribution, particularly in resource-limited countries. To address this issue, we developed a trans-amplifying (TA) mRNA vaccine comprising two mRNAs - one encoding the spike protein of SARS-CoV-2 and the other encoding an RNA-dependent RNA polymerase (from the Venezuelan equine encephalitis virus (VEEV)). This polymerase (also known as replicase), replicates the mRNA coding for the spike protein within host cells post-immunization. The spike protein antigen for the vaccine was derived from the consensus nucleic acid sequence of the SARS-CoV-2 ancestral B.1 lineage and its major variants (alpha, beta, gamma, delta, Omicron BA.1/BA.2, BA.2.12.1, BA.4, and BA.5). The mRNA encoding the spike protein is designed with conserved sequence elements for selective replication by the VEEV replicase. The TA mRNA vaccine induced robust antibody response in Human Angiotensin-converting enzyme-2 (hACE-2)-expressing transgenic mice after two doses of immunization. Notably, the antibody response elicited by TA mRNA vaccine showed broadly cross-neutralizing ability across multiple SARS-CoV-2 variants with only 1/40th of the spike mRNA dosage used in conventional mRNA vaccine. Immunized hACE-2 mice challenged with the SARS-CoV-2 Omicron BA.1 variant showed >10-fold reduction in lung viral titers (by TCID50 and qPCR) at day-4 post-challenge compared to mock-vaccinated controls. SARS-CoV-2 defective viral genomes (DVGs) outcompete the wild-type (WT) virus and thereby act as a potent antiviral therapeutic. We designed RNA mini-genomes based on DVGs comprising specific non-protein-coding segments and representing 10% of the SARS-CoV-2 genome. When transfected into human lung A549 cells infected with SARS-CoV-2 Delta or Omicron BA.1, RNA mini-genome significantly inhibited WT SARS-CoV-2-replication. Co-infection of hACE-2 mice with WT SARS-CoV-2 Omicron BA.1 and RNA-minigenome resulted in a >20-fold reduction in lung viral titers (by TCID50 and qPCR) compared to mice co-infected with WT SARS-CoV-2. In summary, the TA-mRNA vaccine and RNA minigenome are promising broad-spectrum interventions against SARS-CoV-2 warranting further in vivo protection and dose optimization studies.