AbstractImmunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (SPZ) in PfSPZ Vaccine, has provided better vaccine efficacy (VE) against controlled human malaria infection (CHMI) with the same parasites as in the vaccine (homologous) than with genetically distant parasites (heterologous). We sought to identify an immunization regimen that provided similar VE against CHMI with homologous and heterologous Pf for at least 9 weeks in malaria-naïve adults. Such a regimen was identified in part 1 (optimization), an open label study, and confirmed in part 2 (verification), a randomized, double-blind, placebo-controlled study in which VE was assessed by cross-over repeat CHMI with homologous (PfNF54) and heterologous (Pf7G8) PfSPZ at 3 and 9–10 weeks. VE was calculated using Bayesian generalized linear regression. In part 1, vaccination with 9 × 10⁵ PfSPZ on days 1, 8, and 29 protected 5/5 (100%) subjects against homologous CHMI at 3 weeks after the last immunization. In part 2, the same 3-dose regimen protected 5/6 subjects (83%) against heterologous CHMI at both 3 and 9–10 weeks after the last immunization. Overall VE was 78% (95% predictive interval: 57–92%), and against heterologous and homologous was 79% (95% PI: 54–95%) and 77% (95% PI: 50–95%) respectively. PfSPZ Vaccine was safe and well tolerated. A 4-week, 3-dose regimen of PfSPZ Vaccine provided similar VE for 9–10 weeks against homologous and heterologous CHMI. The trial is registered with ClinicalTrials.gov, NCT02704533.