Malaria remains a significant global health problem, with *P. vivax* increasingly prevalent in the Asia-Pacific region. Current efforts to control malaria rely heavily on understanding protective immunity and translating this knowledge into effective vaccines. The chemoprophylaxis and sporozoite (CPS) approach, used successfully with *P. falciparum*, involves repeated low-dose sporozoite exposure with chloroquine to prevent blood-stage malaria. This approach has induced high levels of protection but requires multiple doses. This research explores a novel single-shot vaccination strategy, termed 'hypnoboost', which utilizes the natural relapsing characteristic of *P. vivax* to provide a self-boosting effect. The study hypothesizes that limited blood-stage exposure, following a single high-dose sporozoite vaccination, could augment the immune response against liver-stage parasites, thereby achieving sterile protection.

Existing literature highlights the success of the CPS approach in *P. falciparum* vaccination, suggesting that preventing blood-stage infection is crucial for inducing sterile protection. However, the efficacy of this approach in relapsing malaria, such as *P. vivax*, has not been fully investigated. Studies in monkeys have shown some success in inducing liver-stage immunity with *P. vivax* vaccination, but protection rates have been relatively low. The current study aims to address this gap by testing a novel single-shot vaccination strategy that leverages the relapsing nature of the parasite to provide a self-boosting effect.

Twelve rhesus macaques were divided into three groups: a control group, a CPS-like group (hypnoboost-CPS) receiving chloroquine immediately after vaccination, and a blood-stage-exposed group (hypnoboost-BS) receiving chloroquine after several days of blood-stage infection following each relapse. All animals received a single intravenous injection of 10⁶ *P. cynomolgi* sporozoites. The hypnoboost-BS group was allowed to experience controlled blood-stage parasitemia for several days to evaluate the impact of limited blood-stage exposure on immunity. Following a radical cure with primaquine and chloroquine, animals were challenged with 200 sporozoites. Parasitemia was monitored daily to assess protection. Antibody responses and immune cell subsets were analyzed using ELISA and flow cytometry.

The study found that 75% (3 out of 4) of the animals in the hypnoboost-BS group showed sterile protection against sporozoite challenge. In contrast, only 25% (1 out of 4) of the animals in the hypnoboost-CPS group showed sterile protection. The hypnoboost-BS group experienced controlled blood-stage parasitemia for several days during primary infection and relapses. Despite this exposure, ELISA analysis showed no significant antibody response to blood-stage antigens. Flow cytometry analysis revealed a trend towards increased B-cell frequencies in the hypnoboost-BS group compared to the other groups, but no significant difference was observed in other immune cell populations. A statistical analysis using the log-rank test showed a significant difference between the control and hypnoboost-BS groups (p=0.006).

The results suggest that limited blood-stage exposure after a single high-dose sporozoite vaccination may enhance protective efficacy against relapsing malaria. The 75% sterile protection rate in the hypnoboost-BS group, compared to 25% in the hypnoboost-CPS group, indicates that controlled blood-stage exposure might play a beneficial role in boosting immunity. The absence of significant antibody responses against blood-stage antigens suggests that the protective mechanism may be mediated by other immune components, possibly cellular immunity. Further research is needed to identify the specific immune correlates of protection and to optimize the vaccination dose and adjuvant strategies.

This proof-of-concept study demonstrates that a single-shot, high-dose sporozoite vaccination, combined with controlled blood-stage exposure, may be a viable strategy for developing an effective vaccine against relapsing malaria. Future research should focus on identifying immune correlates of protection, optimizing the vaccination dose and adjuvant selection, and exploring the potential of cross-species protection using genetically attenuated parasites.

The study's limitations include the small sample size (four animals per group) and the use of a non-human primate model. The findings need to be validated in larger studies and ultimately in humans. The lack of significant antibody responses to blood-stage antigens suggests that the protective mechanism might be more complex than initially anticipated, requiring further investigation.