The ANRS VRI06 trial investigated CD40.HIVRI.Env, an Antibody Mediated Vaccine composed by a CD40 monoclonal antibody fused with gp140 Env, designed to target antigen to dendritic cells. Initial results in 72 HIV-uninfected volunteers showed that administration of 0.3-3.0 mg of CD40.HIVRI.Env, given alone or with DNA-HIV-PT123, was safe and immunogenic up to 48 weeks. After week 48 in the trial, 45 volunteers were randomized to receive a late boost (LB) of 0.3 mg CD40.HIVRI.Env, either with or without adjuvant, to assess continued safety and immunogenicity at 2 and 24 weeks post-LB. The median LB interval from VRI06 baseline was 80 and 79 weeks in unadjuvanted (n=23) and adjuvanted (n=22) groups, respectively. Env-specific IgG and functional CD4+ T-cells persisted at the time of the LB, a median of 55 weeks after the last injection at W24. The LB was well tolerated and markedly boosted immune responses. In particular: i) IgG response rates against gp140 and 9 gp120 antigens reached 100% (vaccine-matched) and 82-100% (heterologous) by 24 weeks post-LB; ii) response rates to gp70 V1V2 antigens increased substantially (e.g., from 0-5% to 5-35%) against autologous 96ZM651 and heterologous V1V2 antigens; iii) neutralizing titers against Tier 1 MW965.26 rose from 14-22% to 35-59%; iv) the frequency of polyfunctional Env-specific CD4+ T-cells increased and persisted 24 weeks post-LB. No marked differences were observed between adjuvanted and unadjuvanted LB groups. CD40.HIVRI.Env vaccination is safe and induced broad, potent and long-lasting anti-HIV immune responses recalled by a single late boost, even without adjuvant. Trial registration: Clinical Trials.gov NCT04842682 ||https://www.clinicaltrials.gov/ (2021-04-13)