Study design and participants

In total, 19 malaria-naïve, Duffy blood group positive, adult participants were enrolled and underwent primary blood-stage CHMI with P. vivax (Fig. 1). Out of these, 12 completed secondary homologous CHMI and 2 completed tertiary homologous CHMI. Following review of results from primary and secondary P. vivax CHMI observed in VAC069A–D, the study was amended to study heterologous repeat CHMI with P. falciparum. During VAC069E, 6 participants completed heterologous P. falciparum CHMI: of these, 3 had completed primary CHMI and 3 had completed secondary CHMI with P. vivax during VAC069D. Participants were followed up to three months after each CHMI.

VAC069 was a multicohort study with each cohort (A–E) corresponding to a CHMI. In VAC069A to D, new participants were enrolled to undergo primary CHMI with P. vivax (CHMI-1). In VAC069B to D, participants who had completed CHMI in the previous cohort were invited to undergo secondary homologous CHMI (CHMI-2), followed by tertiary homologous CHMI (CHMI-3) in VAC069C and D. In VAC069E, participants who had previously completed one or two CHMIs with P. vivax were invited to undergo heterologous CHMI with P. falciparum (Pf CHMI). Follow-up (f/u) continued for 3 months after each CHMI. The trial was halted in 2020 due to the COVID-19 pandemic. The time intervals between each CHMI are shown to the left.

The VAC079 study, which was conducted alongside VAC069, enrolled 16 participants to test the efficacy of a protein/adjuvant vaccine targeting P. vivax Duffy-binding protein region II (PvDBPII) by CHMI. A total of 10 participants completed three vaccinations and underwent primary P. vivax CHMI 2–4 weeks after their third vaccination, in parallel with unvaccinated participants in VAC069C (Supplementary Fig. 1A). Five participants who completed primary CHMI received a fourth vaccination followed by secondary homologous P. vivax CHMI 5 months later, in parallel with participants in VAC069D. Safety, immunogenicity and efficacy data from primary CHMI have previously been published²⁰. Here, we include the clinical data from primary and secondary CHMI to support the findings of the VAC069 study.

Demographics and Duffy blood group phenotypes of the participants in the VAC069 and VAC079 studies are shown in Supplementary Table 1. These were comparable across all participants undergoing CHMI, except for a preponderance of females in the VAC079 study. For participants who had Duffy blood group phenotypes Fy(a + b−) or Fy(a − b+), their Duffy genotype was determined. In the VAC069 study, one participant with Duffy phenotype Fy(a + b−) and one with phenotype Fy(a − b+) had an erythroid silent FY*B^ES allele (Duffy genotype FY*A/FY*B^ES and FY*B/FY*B^ES, respectively), which ablates expression of the Fyb antigen in red blood cells and results in half the level of Fy expression²¹.

No serious adverse events (SAEs) deemed related to CHMI occurred in either the VAC069 or VAC079 studies. All participants in both studies remained seronegative for red blood cell alloantibodies post-CHMI, and no seroconversion for HIV, hepatitis B and C, Epstein-Barr Virus (EBV) or Cytomegalovirus (CMV) from baseline was observed.

Parasite multiplication rate is comparable between primary and secondary CHMI

We first asked whether homologous repeat CHMI with the PvW1 clone of P. vivax would lead to a reduction in parasite multiplication rate (PMR). All participants developed blood-stage parasitaemia during primary and secondary CHMI (Fig. 2A, Supplementary Table 4). The time taken to reach the protocol-specified malaria diagnosis threshold and the peak parasitaemia were similar between first, second and third infection (Fig. 2B, Supplementary Fig. 2A, B). There was no significant difference in PMR between primary CHMI (median PMR = 6.4-fold increase per 48 h [range 4.0–11.1]) and secondary CHMI (median PMR = 6.0 [range 3.8–8.4]) (Fig. 2C) and PMR was similar in the two participants who underwent tertiary CHMI (8.0 and 8.7) (Supplementary Fig. 2A). PMR during primary CHMI was similar in individuals with different Duffy blood group phenotypes, although the two participants with an FY*B^ES allele had below average values (Supplementary Fig. 2C). There was no obvious difference in PMR by ethnicity but conclusions are limited by the small number of participants of non-white ethnicity (Supplementary Fig. 2D). In summary, P. vivax CHMI does not generate anti-parasite immunity to slow or reduce parasite growth upon rechallenge. Rechallenge was homologous using the same parasite clone, which removes polymorphism as a possible block to the development of immunity. These results reinforce the notion that anti-parasite immunity is acquired slowly and only after many years of repeated exposure to malaria.

A Parasitaemia was measured up to twice daily by qPCR and is shown for each participant during primary P. vivax (CHMI-1) and secondary P. vivax CHMI (CHMI-2). The mean parasitaemia is shown in bold, and the dashed line indicates the treatment threshold of 10,000 genome copies (gc) ml⁻¹. B Peak parasitaemia for each participant as measured by qPCR during primary and secondary P. vivax CHMI. There was no significant difference between primary and secondary infection (two-tailed p = 0.9, Wilcoxon matched pairs signed-rank test). C Parasite multiplication rate (fold-change per 48 h) was modelled from each participant’s log₁₀-transformed qPCR data. No significant difference was observed between primary and secondary P. vivax CHMI (two-tailed p = 0.2, Wilcoxon matched pairs signed-rank test). In (B, C), box and whisker plots show median and interquartile range (IQR) with whiskers representing 1.5× IQR. All data are shown as dots. D Serum IgG responses were measured to seven P. vivax merozoite antigens using a multiplexed assay: Apical Membrane Antigen 1 (PvAMA1), Duffy-Binding Protein (PvDBP), Erythrocyte Binding Protein (PvEBP), GPI-Anchored Micronemal Antigen (PvGAMA), Merozoite Surface Protein 1 (PvMSP1), 6-Cysteine Protein p12 (PvP12) and Tryptophan Rich Antigen 25 (PvTRAg25). Gene IDs are shown in brackets. Antibody responses to CD4 were measured as a negative control. The following time-points are shown: baseline (1 or 2 days before P. vivax challenge), 7 days after challenge (C + 7), day of diagnosis and 45–56 days after challenge (C + 56). In (A–D), n = 19 (primary CHMI) and n = 12 (secondary CHMI).

Rechallenge leads to boosting of class-switched anti-merozoite antibodies

Anti-parasite immunity is thought to be underpinned by antibody recognition of blood-stage parasite antigens²², and so we assessed whether the comparable rates of parasite growth during primary and secondary P. vivax CHMI were due to a failure to generate class-switched antibody responses. We used a multiplex immunoassay to measure serum IgG against seven P. vivax merozoite antigens: Apical Membrane Antigen 1 (PvAMA1), Duffy-Binding Protein (PvDBP), Erythrocyte Binding Protein (PvEBP), GPI-Anchored Micronemal Antigen (PvGAMA), Merozoite Surface Protein 1 (PvMSP1), 6-Cysteine Protein p12 (PvP12) and Tryptophan Rich Antigen 25 (PvTRAg25) (Fig. 2D). Antibodies specific for PvMSP1 were detectable by 56 days after primary CHMI but responses against other merozoite antigens were either very low in a small number of participants or absent. Antibodies against PvMSP1, PvAMA1, PvDBP and PvTRAg25 were all boosted upon rechallenge and already evident at diagnosis. Circulating antibody titres therefore increased during secondary homologous CHMI, but, given the comparable parasite growth kinetics during primary and repeat CHMI, these antibodies were evidently insufficient to slow parasite growth.

Antibodies targeting region II of PvDBP have the potential to block merozoite invasion by interrupting the crucial interaction between P. vivax and the Duffy antigen expressed on reticulocytes. PvDBPII is the leading blood-stage candidate vaccine antigen, and we previously reported in the VAC079 trial that vaccine-induced responses of 150–340 µg/mL, as measured by ELISA, could reduce P. vivax growth by ~50% following primary PvW1 blood-stage CHMI²⁰. We therefore asked whether IgG antibodies specific for region II of PvDBP were also naturally generated during CHMI. However, IgG levels remained undetectable (2E). On the other hand, we observed a small response to the large 140 kDa full-length PvDBP ectodomain in the multiplex immunoassay after secondary CHMI (Fig. 2D). Class-switched antibodies specific for merozoite antigens are thus generated and boosted upon a single vivax rechallenge but lack the required breadth and/or specificity to effectively neutralise reticulocyte invasion.

A single infection induces long-lived mechanisms of clinical immunity

Clinical immunity can be generated even in the absence of parasite control^13,14 and has been observed after one malaria infection in neurosyphilis patients¹⁷. We therefore assessed whether the frequency or severity of symptoms was altered between the first and second CHMI. In VAC069, at least one solicited adverse event (AE) was reported by all participants during both primary and secondary CHMI. During primary CHMI, solicited AEs increased in frequency and severity around the time of diagnosis, peaked within 48 h and mostly resolved by six days after starting drug treatment (Supplementary Fig. 3A). A minority of participants reported AEs relating to antimalarial drugs, which resolved quickly upon completion of treatment (Supplementary Fig. 3B). Unsolicited AEs assessed to be at least possibly related to primary CHMI were predominantly of mild severity, with decreased appetite the most frequent symptom reported (Supplementary Table 2A). The maximum severity of any solicited AE was markedly reduced during rechallenge, with only 1 out of 12 (8%) participants undergoing secondary CHMI reporting at least one severe (grade 3) AE, compared to 9 out of 19 (47%) participants undergoing primary CHMI (Fig. 3A). The most commonly reported solicited AEs were headache, fatigue and malaise. All solicited AEs occurred less frequently and with lower severity during secondary compared to primary CHMI (Fig. 3B).

A, B Clinical signs and symptoms of malaria in participants undergoing primary P. vivax CHMI (CHMI-1) compared to secondary P. vivax CHMI (CHMI-2) in the VAC069 study. Data are shown as a proportion of the total number of participants undergoing CHMI. A shows the maximum severity of any solicited adverse event (AE) reported by an individual in the 48 h before and after diagnosis; (B) shows the frequency and severity of each solicited AE. C Maximum recorded temperature during primary and secondary P. vivax CHMI. The pink dots represent the one participant who experienced fever upon rechallenge. All data shown, statistical comparison using two-tailed Wilcoxon matched pairs signed-rank test (p = 0.0099) only for participants undergoing two CHMIs (n = 12). D–H Laboratory parameters (biochemistry and full blood counts) were measured during primary and secondary P. vivax CHMI at baseline (1 or 2 days before challenge); 7 and 14 days after challenge (C + 7 and C + 14); on the day of diagnosis; 1, 3 and 6 days after treatment (T + 1, T + 3 and T + 6); 45 to 56 days (C + 56) and 96 days (C + 96) after challenge. Statistically significant differences between CHMI-1 and CHMI-2 were identified at each time-point by using mixed-effects modelling and linear regression. D shows alanine aminotransferase (ALT, T + 6 p = 1.1 × 10⁻⁷); (E) shows albumin (diagnosis p = 0.039, T + 1 p = 1.3 × 10⁻⁵, T + 3 p = 8.6 × 10⁻⁷, T + 6 p = 1.3 × 10⁻⁶). F shows the minimum haemoglobin concentration with data split by participant sex. There was no significant difference between CHMI-1 and CHMI-2 (Wilcoxon matched pairs signed-rank test, two-tailed p = 0.8 for females, p = 0.09 for males). G shows lymphocyte count (diagnosis and T + 1 p −16, T + 6 p = 0.029); (H) shows platelet count (diagnosis p = 3.5 × 10⁻⁸, T + 1 p −16, T + 3 p = 4.1 × 10⁻¹⁰). Box and whisker plots show median and interquartile range (IQR) with whiskers representing 1.5× IQR (outliers are shown as dots). In (A–F), n = 19 (primary P. vivax CHMI) and n = 12 (secondary P. vivax CHMI).

Symptoms reported by participants are subjective, so we assessed whether there was a comparable reduction in the frequency and severity of quantifiable clinical and laboratory parameters (Supplementary Table 3A). Pyrexia (temperature >37.5 °C) was recorded in 17 out of 19 (89%) participants during primary CHMI but in only 1 out of 12 participants (8%) upon rechallenge. Consequently, the maximum recorded temperature was significantly lower in secondary CHMI (median = 37.2 °C) compared to the same 12 participants during primary CHMI (median = 38.3 °C, Wilcoxon signed-rank test p 3C). Raised alanine aminotransferase (ALT) (>55 IU L⁻¹) was observed in 12 out of 19 (63%) participants undergoing primary CHMI, with ALT levels peaking around six days post-treatment and normalising by 2 months after challenge (Fig. 3D). This was severe in three participants with the highest recorded ALT at 14 times the upper limit of normal. All participants with raised ALT had normal alkaline phosphatase (ALP) and bilirubin levels, and clotting tests performed in those with severe transaminitis also remained normal. ALT at day 6 post-treatment was significantly lower during secondary compared to primary CHMI, and transaminitis was only observed in 1 out of 12 (8%) participants during secondary CHMI. Of note, this individual continued to have significant malaria symptoms and was the only participant who was febrile upon both secondary and tertiary rechallenge (Fig. 3C). Serum albumin was also significantly lower at days 1–6 post-treatment during primary CHMI compared to during rechallenge (Fig. 3E).

We observed a slight reduction in haemoglobin 6 days after starting antimalarial treatment, but these did not differ between primary and secondary CHMI (Fig. 3F). In contrast, lymphopaenia was pronounced, reaching a nadir at 24 h after diagnosis and was significantly attenuated upon rechallenge (linear regression p 3G). Thrombocytopaenia, also lowest at 24 h after treatment, was similarly significantly attenuated during secondary compared to primary CHMI (linear regression p 3H). Taken together, these data indicate that participants are protected against pyrexia, liver injury, lymphopaenia and thrombocytopaenia when undergoing homologous repeat P. vivax CHMI. In concordance with these results, we also observed reduced signs and symptoms of malaria after rechallenge in the VAC079 study. In the VAC079 study, the rate of fever and the severity of solicited AEs and laboratory abnormalities such as transaminitis were similar to VAC069 during primary CHMI but were significantly reduced during secondary CHMI (Supplementary Table 3B, Supplementary Fig. 1B–D). Unsolicited AEs, which were mostly mild in severity, were also reported less frequently during secondary compared to primary CHMI (Supplementary Table 2B). A single infection with P. vivax is therefore sufficient to generate and maintain mechanisms of long-lived clinical immunity (20 months in this study) that can reduce the harm caused by malaria parasites during subsequent infections, and these mechanisms are independent of anti-parasite immunity.

Clinical immunity is underpinned by attenuated inflammation

Many of the symptoms of malaria, such as fever and laboratory abnormalities, including lymphopaenia, are caused by the host response to infection⁶. We hypothesised that systemic inflammation would be attenuated upon rechallenge to raise the pyrogenic threshold and improve clinical outcome. We therefore measured plasma analytes indicative of inflammation, coagulation, oxidative stress and tissue damage using a bead-based multiplexed protein assay and compared the dynamics of each analyte through time during primary and secondary CHMI. For these experiments, we selected 7 participants who underwent primary CHMI in VAC069C and secondary CHMI in VAC069D, as well as 3 participants who underwent primary CHMI in VAC069D. Samples were not used from VAC069A or VAC069B because blood samples were not collected at all post-treatment time-points.

We found that plasma levels of the major pyrogenic cytokines or their regulators interleukin 1 receptor A (IL-1RA), interleukin 6 (IL-6) and soluble tumour necrosis factor receptor 2 (sTNFRII) were raised at diagnosis and peaked 24 h after drug treatment during primary CHMI (Fig. 4A). However, these inflammatory markers were significantly reduced upon rechallenge. The interferon-stimulated C-X-C motif chemokine ligand 10 (CXCL10), the critical host factor for recruitment of T cells into inflamed tissues, as well as the cytokines IL-12p70 and IL-18, which promote T cell activation and differentiation, were also attenuated during rechallenge (Fig. 4B). Similarly, markers of coagulation and endothelium activation, which peaked between 1 and 3 days after diagnosis in primary CHMI, were reduced upon rechallenge and remained at almost baseline levels during and after secondary CHMI (Fig. 4C). Our data reveal that P. vivax can rapidly induce host adaptations that restrict inflammation, avert a pro-coagulant state and prevent endothelial dysfunction to raise the pyrogenic threshold and minimise the clinical symptoms associated with malaria.

A–C Circulating biomarkers of inflammation, coagulation and endothelial cell activation were quantified during and after primary and secondary P. vivax CHMI using a bead-based multiplexed protein assay. We analysed plasma proteins at baseline (1 or 2 days before challenge); 12 days after challenge (C + 12); on the day of diagnosis; 1, 3 and 6 days after treatment (T + 1, T + 3 and T + 6); and 45 to 56 days after challenge (C + 56). A shows pyrogenic cytokines and their regulators (IL-1Ra: diagnosis p = 0.0016, T + 1 p = 6.8 × 10⁻¹¹, IL-6: diagnosis p = 0.0024, T + 1 p = 2.1 × 10⁻⁷, sTNFRII: diagnosis p = 4.1 × 10⁻⁷, T + 1 p −16, T + 3 p = 8.5 × 10⁻⁵). (B) shows chemokines and cytokines involved in T cell recruitment and activation (CXCL10: T + 1 p = 3.9 × 10⁻⁵, IL-12p70: diagnosis p = 0.0033, T + 1 p = 0.0048, IL-18: diagnosis p = 6.6 × 10⁻⁴, T + 1 p = 4.2 × 10⁻¹⁵, T + 3 p −16, T + 6 p = 6.0 × 10⁻¹³) and (C) shows markers of coagulation and endothelial cell activation (D-Dimer: T + 1 p = 2.1 × 10⁻⁸, E-selectin: diagnosis p = 0.0038, T + 1 p = 1.2 × 10⁻⁷, T + 3 p = 2.4 × 10⁻⁵, T + 6 p = 0.014, ICAM-1: diagnosis p = 3.6 × 10⁻⁴, T + 1 p = 2.9 × 10⁻¹⁰, T + 3 p = 4.8 × 10⁻⁹, T + 6 p = 7.6 × 10⁻⁵). Box and whisker plots show median and interquartile range (IQR) with whiskers representing 1.5× IQR (outliers are shown as dots). Statistically significant differences between CHMI-1 and CHMI-2 were identified at each time-point by using mixed-effects modelling and linear regression. In (A–C), n = 10 (primary P. vivax CHMI) and n = 7 (secondary P. vivax CHMI).

Clinical immunity is parasite species-specific

Given that P. vivax is co-endemic with P. falciparum across much of the world^2,3,4, we next wanted to assess whether clinical immunity is specific to the parasite species that raised the pyrogenic threshold. Neurosyphilis patients were sometimes infected with P. falciparum once they became refractory to P. vivax-induced fever²³. We therefore hypothesised that clinical immunity, which developed against P. vivax, would not be effective against rechallenge with P. falciparum. To test this hypothesis, we amended VAC069E to infect participants who had previously undergone one or two prior P. vivax CHMIs, with the 3D7 clone of P. falciparum using a blood challenge²⁴. P. falciparum, which preferentially invades mature red cells, has a higher PMR than P. vivax, which is restricted to reticulocytes. In concordance with this, we found that in VAC069E, participants infected with P. falciparum reached the treatment threshold quicker than when previously infected with P. vivax (Fig. 5A). Peak parasitaemia was comparable to primary and secondary P. vivax CHMI, therefore any differences between homologous and heterologous rechallenge in the VAC069 study were not confounded by circulating pathogen load. P. falciparum growth dynamics and the time to reach malaria diagnostic criteria were also similar to those seen in primary blood-stage P. falciparum CHMI in our previous studies, VAC054 and VAC063, conducted in Oxford using the same 3D7 clone^24,25,26 (Supplementary Fig. 4A, B).

A Parasitaemia as measured by qPCR during primary (CHMI-1) and secondary P. vivax CHMI (CHMI-2) and heterologous rechallenge with P. falciparum. Mean parasitaemia is shown in bold. The dashed line indicates the treatment threshold of 10,000 genome copies (gc) ml⁻¹. B shows the maximum severity of any solicited adverse event (AE) reported by an individual in the 48 h before and after diagnosis, as a proportion of the total number of participants, during primary and secondary P. vivax CHMI and heterologous P. falciparum rechallenge. C Heatmap showing the log₂ fold-change of 24 plasma analytes during primary and secondary P. vivax CHMI and heterologous P. falciparum rechallenge. Data are shown relative to values at baseline (1 or 2 days before challenge) at the following time-points: 7 (C + 7) or 12 (C + 12) days after challenge with P. falciparum or P. vivax, respectively; day of diagnosis; 1, 3 and 6 days after treatment (T + 1, T + 3, T + 6); and 45 to 56 days after challenge (C + 56). Analytes are ordered by unsupervised hierarchical clustering, and those that vary significantly between primary and secondary P. vivax CHMI and between secondary P. vivax CHMI and P. falciparum rechallenge are indicated in green; grey is non-significant. Significance was assessed using mixed-effects modelling and linear regression. The data for each CHMI are paired. D–G Laboratory parameters were measured during primary and secondary P. vivax CHMI and heterologous P. falciparum rechallenge, at the same time-points as in (C), as well as at 96 days (C + 96) after challenge. D shows lymphocyte count (no significant difference between first vivax and heterologous falciparum rechallenge for minimum lymphocyte counts (p = 0.97 by two-tailed Wilcoxon matched pairs signed-rank test); (E) shows platelet count (diagnosis p = 2.0 × 10⁻⁹, T + 1 and T + 3 p −16); (F) shows alanine aminotransferase (ALT, T + 6 p = 5.1 × 10⁻⁹); (G) shows albumin (T + 1 p = 3.7 × 10⁻⁵, T + 3 p = 4.0 × 10⁻⁹, T + 6 p = 9.7 × 10⁻¹⁰) p = 0.0084. Box and whisker plots show median and interquartile range (IQR) with whiskers representing 1.5× IQR (outliers are shown as dots). In (E–G), statistically significant differences between primary P. vivax CHMI and heterologous P. falciparum rechallenge were identified at each time-point using mixed-effects modelling and linear regression. In (A, B) and (D–G), n = 19 (primary P. vivax CHMI), n = 12 (secondary P. vivax CHMI) and n = 6 (heterologous P. falciparum CHMI). In (C) n = 10 (primary P. vivax CHMI), n = 7 (secondary P. vivax CHMI) and n = 6 (heterologous P. falciparum CHMI).

Clinical symptoms during VAC069E occurred at a similar frequency and severity to primary P. vivax CHMI (Fig. 5B, Supplementary Table 2C, Supplementary Table 3C) and were comparable to primary P. falciparum infections in our previous studies, P. falciparum CHMI studies VAC054 and VAC063 (Supplementary Fig. 4C)^24,25,26. The proportion of participants experiencing fever in VAC069E (2 out of 6 [33%] participants) did not significantly deviate from that observed during primary CHMI in the VAC054 and VAC063 studies (23 out of 39 [59%] participants)^24,25,26 (Barnards CSM exact test p = 0.3). During VAC069E, the inflammatory and coagulation markers that were attenuated when participants underwent secondary P. vivax CHMI (including IL-6, CXCL10 and D-Dimer) were all significantly increased again during heterologous P. falciparum rechallenge (Fig. 5C). Heterologous P. falciparum rechallenge led to a pronounced lymphopaenia that was comparable to primary P. vivax CHMI (Fig. 5D), whereas thrombocytopaenia and liver injury remained attenuated during heterologous P. falciparum rechallenge compared to primary P. vivax CHMI (Fig. 5E–G). When compared to primary P. falciparum CHMI in the VAC054 and VAC063 studies, lymphopaenia and thrombocytopaenia were comparable in those undergoing heterologous P. falciparum rechallenge in VAC069E (Supplementary Fig. 4D, E). In our historical studies VAC054 and VAC063, no blood tests were taken at day 6 post-treatment, at the peak of ALT rise, so we cannot compare rates of transaminitis to primary P. falciparum CHMI. Our data show that infection with P. falciparum after prior exposure to P. vivax leads to systemic inflammation, fever and lymphopenia comparable to that observed during primary P. vivax and primary P. falciparum CHMI. Clinical immunity, as defined by symptomatology, is thus parasite species-specific.