Background: Phase IIb HIPRA-HH-2 study results showed that PHH-1V as first booster dose elicited a strong and sustained neutralising antibody response against various SARS-CoV-2 variants. Here, we report the safety and immunogenicity of a fourth booster dose of PHH-1V against the most prevalent Omicron SARS-CoV-2 variants in Spain. Methods: The HIPRA-HH-2 open-label extension study (NCT05142553) evaluated the safety and immunogenicity of PHH-1V as a fourth booster dose in subjects aged ≥18 years and followed for 6 months. Subjects received a fourth dose of PHH-1V 6-12 months after a previous regime of either two doses of BNT162b2 plus a third dose of PHH-1V (Cohort 1) or three doses of BNT162b2 (Cohort 2). Primary regulatory endpoint evaluated the neutralisation titres (GMT) against Omicron BA.1 on Day 14 of PHH-1V used as fourth dose in Cohort 2 vs the BNT162b2 used as third dose in initial HIPRA-HH-2 study. The immunogenicity of PHH-1V as fourth dose was also investigated by GMTs against Beta, Delta, and Omicron BA.1, BA.4/5 and XBB.1.5 on Days 14, 98 and 182 post-immunisation in the overall study population and in Cohorts 1 and 2 versus baseline. Safety of the fourth dose was also assessed. Findings: From September 2022, 288 subjects received PHH-1V as a fourth dose (Cohort 1 n=106; Cohort 2 n=182). A significant increase in neutralising antibodies against Omicron BA.1 subvariant at Day 14 was observed from the third homologous booster with mRNA vaccine compared to the fourth heterologous booster with PHH-1V (1739.02 vs 4049.01; GMT ratio 0.43 (95% CI: 0.28; 0.65; p-value < 0.0001). PHH-1V used as fourth booster induced a statistically significant increase in neutralising antibody titres 14 days after immunisation for all variants compared with baseline [GMFR on Day 14 (95%CI) was 6.96 (5.23, 9.25) for Beta variant; 6.27 (4.79, 8.22) for Delta variant; 9.21 (5.57, 15.21) for Omicron BA.1 variant; 11.80 (8.29, 16.80) for Omicron BA.4/5 variant and 5.22 (3.97, 6.87) for Omicron XBB.1.5 variant]. Titres remained significantly higher compared with baseline at 3 and 6 months post-vaccination. Cohort comparison revealed no significant differences at 14 , 98 and 182 days post-vaccination. The most frequent adverse events were injection site pain (Cohort 1: 84.0%; Cohort 2: 77.5%) and fatigue (Cohort 1: 17.9%; Cohort 2: 29.1%). No subjects experienced severe COVID-19 infection. Interpretation: The PHH-1V vaccine as a booster induced a potent and sustained neutralising antibody response against previous circulating Beta, Delta variants and Omicron BA.1, BA.4/5, and XBB.1.5 subvariants in subjects previously vaccinated with three doses regardless of previous regimen. These findings suggest that PHH-1V could be an appropriate strategy for upcoming heterologous vaccination campaigns.