Introduction:
Human T-cell Leukemia Virus type 1 (HTLV-1) retrovirus is estimated to infect 10 million people worldwide. Infection is lifelong and around 5% of infected individuals will develop severe diseases, such as adult T-cell leukemia (ATL). Approximately 37% of people in First Nations communities in Central Australia are infected with HTLV-1 subtype C. To date, no curative interventions or prophylactic vaccines exist. Recent success with vaccine control of Hepatitis B using virus-like particles (VLP) and with mRNA-vaccines expressing SARS-CoV-2 stabilized-Spike introduces promising new approaches. This study aims to develop mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing VLPs coated with prefusion-stabilized HTLV-1 Envelope glycoprotein (Env), to elicit protective host immune-responses against HTLV-1.
Methods:
In vitro transcription (IVT) of mRNA expressing HTLV-1 VLP backbone constructs (Gag polyprotein) and Env constructs were transfected or co-transfected into HEK293T cells to produce VLPs. Ultracentrifuge-purified VLP samples and cell lysates were loaded onto Western blot for protein characterization. VLP morphology was validated by electron microscopy and Env expression was tracked by Confocal microscopy.
Results:
Multiple versions of HTLV-1 VLP backbone and Env constructs were developed as RNA-production vector templates. Codon-optimized mRNA with Clean-Cap and N-1-methyl-psudouridine was produced by IVT. Immunoblot analysis confirmed intracellular Gag and Env expression. However, confocal microscopy showed VLP did not incorporate Env which was also missing from the cell surface. Further mutation is underway to enhance expression onto the cell surface.
Discussion:
VLPs were successfully generated via transfection of IVT mRNA, but we observed low-levels incorporation of membrane-anchored HTLV-1 Env. Other published studies document similar issues with various viral glycoproteins. VLP construct optimization for efficient expression of stabilized-trimeric Env is critical for protective vaccine efficiency. Pre-clinical testing in mice of our vaccine prototypes will soon test for the protective immune responses needed to prevent HTLV-1 infection or disease in endemic regions.