SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptorsin vitro. Alhydrogel®‘85’-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates excellent safety profile in repeat-dose GLP toxicity study. In summary, subcutaneous prime/boost DYAI-100A85 inoculation induces high titers of RBD-specific neutralizing antibodies and protection of hACE2-transgenic mice against lethal challenge with SARS-CoV-2. Given its demonstrated safety, efficacy, and low production cost, vaccine candidate DYAI-100 received regulatory approval to initiate a Phase 1 clinical trial to demonstrate its safety and efficacy in humans.