Participant demographics
One hundred thirty-five volunteers were screened for eligibility and 80 were randomized and vaccinated between 20 July 2022 and 27 December 2022. All completed scheduled vaccinations and the first cohort (that is, n = 40) were eligible for CHMI undertaken from 27 November 2022 to 17 December 2022. Three did not proceed to CHMI owing to SARS-CoV-2 positive results (n = 2) and increase in liver enzymes (alanine aminotransferase (ALT), n = 1), so that 37 completed CHMI (Fig. 1). One volunteer was qPCR positive for malaria parasites before vaccination, and all were qPCR negative for malaria parasites before CHMI. There was a predominance of young male volunteers (28/40, 70%) with a mean age of 28.3 years (Table 1). Volunteers were from the Mijikenda ethnic group. The male predominance among volunteers appears to relate to established gender roles26.
Out of 135 volunteers screened, 80 volunteers who were eligible and met the enrollment criteria were randomized into one of four groups across two enrollment cohorts 4 weeks apart. Of the 55 excluded volunteers, those with abnormal laboratory results included low hemoglobin levels (<10 g dl−1 for females (n = 6)), thrombocytopenia (n = 2) and elevated levels of ALTs (n = 1). Other exclusions included being eligible but did not turn up for screening results (n = 4), not being on any method of contraception (n = 3), number required attained (n = 3), did not meet location of residence criteria (n = 2) and did not attend enrollment visit (n = 2). Other reasons, all with a frequency of one (n = 8) were written informed consent not signed, unstable blood pressure values, positive pregnancy test, abdominal hernia, neurofibromatosis, allergy to ibuprofen, failed test of understanding and history of schizophrenia. Seven volunteers had more than one screening failure and these were elevated laboratory values (n = 4), abdominal mass (n = 1), abnormal electrocardiogram (ECG) (n = 1) and substance use (n = 1). First dose, second dose and third dose indicate the respective vaccine doses.
The prespecified primary endpoint was time to meeting treatment criteria (that is, reaching the parasitaemia threshold of 500 parasites μl−1 or any parasitaemia plus important clinical symptoms). Prespecified secondary endpoints were (1) immunology (specifically antibody responses to CSP measured by enzyme-linked immunosorbent assay (ELISA) and T cell responses to ME-TRAP peptides measured by ELISPOT) and (2) efficacy (time to parasitemia >20, >500, >1,000 and >10,000, respectively). All these prespecified outcomes are reported here and are not being published elsewhere.
Primary outcomes
CHMI was conducted 28 days after the last vaccination. All 8 unvaccinated control volunteers and all 12 volunteers vaccinated with ME-TRAP became PCR positive for malaria parasites following ID challenge with cryopreserved sporozoites produced by Sanaria (PfSPZ), with observed typical parasite growth curves on qPCR. Seven of eight control volunteers (88%) and 11 of 12 ME-TRAP volunteers (92%) met the criteria for the primary endpoint and received malaria treatment during CHMI, and one control and one ME-TRAP volunteer remained just below the parasitaemia threshold and so did not meet the primary endpoint (Fig. 2a,c and Table 2).
a–e, The qPCR results (y axis, log transformed) by time after inoculation (x axis) showing ID challenge with PfSPZ, control group (n = 8) (a); ID challenge with PfSPZ, vaccinated with R21/Matrix-M (n = 12) (b); ID challenge with PfSPZ, vaccinated with viral vectors encoding ME-TRAP (n = 12) (c); DVI challenge with PfSPZ, control group (n = 34) (d); and DVI challenge with PfSPZ, vaccinated with R21/Matrix-M (n = 5). Parasitemia was determined by asexual 18S ribosomal RNA gene qPCR done in Kilifi. The blue lines represent individuals who reached the required malaria diagnosis criteria, green lines represent individuals who did not meet the criteria for diagnosis but were qPCR positive, orange lines represent individuals who were qPCR negative throughout monitoring and red dots denote individuals who were febrile based on the criteria for diagnosis.
In contrast, none of the 12 volunteers vaccinated with R21 met the primary endpoint following challenge with ID PfSPZ. Three out of the nine volunteers were briefly positive by PCR for malaria parasites but with no evidence of parasite growth. Hence they were protected and did not meet the primary endpoint (Fig. 2b and Table 1).
Historically unvaccinated control volunteers who had previously undergone CHMI with PfSPZ challenge by DVI showed similar growth rates to PfSPZ challenge ID volunteers, with 31 out of 34 (91%) volunteers meeting the primary endpoint, 2 out of 34 being PCR negative (6%) and 1 out of 34 being PCR positive but not meeting the primary endpoint (3%) (Fig. 2d). However, when the five volunteers vaccinated with R21 underwent CHMI with PfSPZ challenge by DVI sporozoites, all five showed typical growth curves of parasites and met the primary endpoint (Fig. 2e).
Hence, none of the volunteers vaccinated by R21 who received the ID PfSPZ challenge met a primary endpoint, whereas volunteers for all other groups met primary endpoints between days 12 and 21 (P < 0.0005 by log-rank survival across all groups; Fig. 3). Parasites were genotyped for AMA1, confirming the challenge parasite strain (NF54) in all cases.
Kaplan–Meier curve of the fraction of volunteers that did not meet the primary efficacy endpoint criteria for treatment (that is, 500 parasites per µl, or any parasite density plus clinically significant symptoms) following CHMI. P < 0.0005 by log-rank testing across all groups.
Secondary outcomes
Immunogenicity outcomes were the secondary outcomes. Following the vaccination course with R21, anti-NANP antibodies rose from baseline levels of just under 10 ELISA units (EU) to above 1,000 EU in the R21-vaccinated volunteers, but remained below 10 among the control group (Extended Data Fig. 1). Peak responses were geometric means of 2,152 and 1,113 and minimum to maximum ranges of 750–5,500 versus 610–3,300 for ID versus DVI challenged groups, respectively (P = 0.14).
Following vaccination with viral vectors encoding ME-TRAP, spot-forming units (s.f.u.) per million peripheral blood mononuclear cells (PBMCs) increased from geometric mean of 104 s.f.u. (95% CI 70–153) at baseline to a peak of 735 (95% CI 364–1,485 s.f.u., P = 0.01 for both comparisons with baseline) and 335 s.f.u. (95% CI 162–691 s.f.u.) on the day of challenge, compared with 127 s.f.u. (95% CI 74–409 s.f.u.) among the control group; P = 0.064, P = 0.001 and P = 0.084 for comparisons between all controls and ME-TRAP vaccinated volunteers at days 0, 63 and 84, respectively (Extended Data Fig. 2).
Volunteers had evidence of past exposure to malaria, as evidenced by prevaccination IgG antibodies against schizont extract (Extended Data Fig. 3). Historical controls had higher titers than the newly recruited cohort (geometric means of 1,244,000 EU (95% CI 693,000–2,235,000 EU) versus 394,000 EU (95% CI 258,000–602,000 EU), respectively, P = 0.0009), and the DVI and ID challenged groups had similar schizont extract antibodies (geometric means of 284,000 EU (95% CI 158,000–511,000 EU) versus 434,000 EU (95% CI 252,000–745,000 EU), respectively, P = 0.4).
Secondary efficacy outcomes were consistent with the primary outcome, that is, none of the volunteers vaccinated by R21 who received the ID PfSPZ challenge met the secondary endpoints, whereas volunteers for all other groups met secondary endpoints between days 7 and 21, none of the 12 volunteers in the R21 ID group met any of the secondary endpoints, whereas 75/78 (96.1%), 60/78 (76.9%), 56/78 (71.8%) and 39/78 (50%) met secondary endpoints (P < 0.0005, P = 0.0001, P = 0.0007 and P < 0.0005 by log-rank survival across all groups for time to parasitemia >20, >500, >1,000 and >10,000, respectively; Extended Data Fig. 4).
Safety
Following vaccination, self-limiting local solicited adverse events and general adverse events such as headache or fatigue were detected in a minority of volunteers (Table 3). No event was reported as severe and four events were reported as moderate, with a median and maximum duration of 4.5 and 5 days, respectively. General adverse events were similar in the R21 and ME-TRAP groups after the first vaccination. Specifically, headache occurred in 0/24, 1/14 (7.1%) and 5/24 (21%); fatigue in 2/24 (8.3%), 0/14 and 3/24 (12%); and malaise in 2/24 (8.3%), 0/14 and 2/24 (8.3%) of volunteers allocated to the R21 ID, R21 DVI and ME-TRAP ID groups, respectively. Fever was a more common general adverse event at the final vaccination for ME-TRAP with 5/24 (20.8%) and 8/24 (33.3%) experiencing headache, but only 1/38 volunteers vaccinated with R21 experienced fever (2.6%) and 2/38 experienced headache (5.2%). Local adverse events were rare and limited to pain at the injection site in 1/24 (4.2%) and 2/24 (8.3%) in the R21 ID and ME-TRAP ID groups, respectively.
During CHMI, there were no immediate or early adverse events, but after day 7, fever, headache and fatigue were common in 22/37 (59.5%), 17/37 (45.9%) and 12/37 (32.0%) of volunteers, respectively (Extended Data Table 1). Six events were reported to be moderate and none were reported as severe. All events resolved on follow-up.
Abnormal blood tests, primarily transient elevations in liver enzymes and/or low white blood counts were identified in several volunteers but were not clinically significant and resolved on follow-up (Extended Data Table 2). There were clinically nonsignificant minor elevations in liver enzymes among the R21 ID group, and during CHMI there were clinically nonsignificant, transient abnormalities of liver enzymes and platelet counts (Extended Data Table 3). The safety findings were consistent with those reported previously in the same population27.
Sensitivity analysis
We saw one-off PCR positive signals on a single time point among 3 of the 12 volunteers (25%) vaccinated with R21 who received PfSPZ Challenge by ID If we include these volunteers as meeting an exploratory endpoint for purposes of a sensitivity analysis, protection is nevertheless substantial by ID (9 out of 12, 75%), and statistically significantly different from DVI (0 out of 5 protected, P = 0.009 by Fisher’s two-sided test).
Post hoc analysis
The geometric mean parasite densities by PCR were similar over time following PfSPZ challenge for the control group by DVI, the control group by ID challenge, ME-TRAP vaccines by ID challenge and R21 vaccines by DVI (Extended Data Fig. 5). Hence, parasite inoculum and growth rates appear similar among these four groups.
