Access to vaccines against SARS-CoV-2 virus was limited in poor countries during the COVID-19 pandemic. Therefore, a low-cost mRNA vaccine, PTX-COVID19-B, was produced and evaluated in a Phase 1 trial. PTX-COVID19-B encodes Spike protein D614G variant without the proline-proline (986–987) mutation present in other COVID-19 vaccines. The aim of the study was to evaluate safety, tolerability, and immunogenicity of PTX-COVID19-B vaccine in healthy seronegative adults 18–64 years old. The trial design was observer-blinded, randomized, placebo-controlled, and tested ascending doses of 16-µg, 40-µg, or 100-µg in a total of 60 subjects who received two intramuscular doses, 4 weeks apart. Participants were monitored for solicited and unsolicited adverse events after vaccination and were provided with a Diary Card and thermometer to report any reactogenicity during the trial. Blood samples were collected on baseline, days 8, 28, 42, 90, and 180 for serum analysis of total IgG anti-receptor binding domain (RBD)/Spike titers by ELISA, and neutralizing antibody titers by pseudovirus assay. Titers in BAU/mL were reported as geometric mean and 95% CI per cohort. After vaccination, few solicited adverse events were observed and were mild to moderate and self-resolved within 48 h. The most common solicited local and systemic adverse event was pain at the injection site, and headache, respectively. Seroconversion was observed in all vaccinated participants, who showed high antibody titers against RBD, Spike, and neutralizing activity against the Wuhan strain. Neutralizing antibody titers were also detected against Alpha, Beta, and Delta variants of concerns in a dose dependent manner. All tested doses of PTX-COVID19-B were safe, well-tolerated, and provided a strong immunogenicity response. The 40-µg dose showed fewer adverse reactions than the 100-µg dose, and therefore was selected for a Phase 2 trial, which is currently ongoing.Clinical Trial Registration number: NCT04765436 (21/02/2021). (https://clinicaltrials.gov/ct2/show/NCT04765436).