Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies

BACKGROUND. Plasmodium vivax is the most widespread human malaria geographically; however, no effective vaccine exists. Red blood cell invasion by the P. vivax merozoite depends on an interaction between the Duffy antigen receptor for chemokines (DARC) and region II of the parasite’s Duffy-binding protein (PvDBP_RII). Naturally acquired binding-inhibitory antibodies against this interaction associate with clinical immunity, but it is unknown whether these responses can be induced by human vaccination. METHODS. Safety and immunogenicity of replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and modified vaccinia virus Ankara (MVA) viral vectored vaccines targeting PvDBP_RII (Salvador I strain) were assessed in an open-label dose-escalation phase Ia study in 24 healthy UK adults. Vaccines were delivered by the intramuscular route in a ChAd63-MVA heterologous prime-boost regimen using an 8-week interval. RESULTS. Both vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. PvDBP_RII–specific ex-vivo IFN-γ T cell, antibody-secreting cell, memory B cell, and serum IgG responses were observed after the MVA boost immunization. Vaccine-induced antibodies inhibited the binding of vaccine homologous and heterologous variants of recombinant PvDBP_RII to the DARC receptor, with median 50% binding-inhibition titers greater than 1:100. CONCLUSION. We have demonstrated for the first time to our knowledge that strain-transcending antibodies can be induced against the PvDBP_RII antigen by vaccination in humans. These vaccine candidates warrant further clinical evaluation of efficacy against the blood-stage P. vivax parasite. TRIAL REGISTRATION. Clinicaltrials.gov NCT01816113. FUNDING. Support was provided by the UK Medical Research Council, UK National Institute of Health Research Oxford Biomedical Research Centre, and the Wellcome Trust.


Introduction
Five species of Plasmodium parasite are known to cause malaria following human infection, with P. falciparum the major causative agent of deaths in sub-Saharan Africa and thus historically the dominant focus of vaccine development efforts (1). However, a second parasite species, P. vivax, is more widespread geographically and also constitutes a significant proportion of human malaria cases. Indeed, recent data suggest 2.5 vectored vaccines to deliver protein antigens of interest with the key aim of inducing antibodies in conjunction with T cell responses. The most successful approach to date has utilized a recombinant replication-deficient adenovirus (of human or simian serotype) to prime the immune response, followed by a booster vaccination (typically 8 weeks later) with an attenuated poxvirus recombinant for the same antigen (32). These vectors have shown high-titer antibody induction against numerous difficult-to-express malaria antigens in animal models, including NHPs (33,34). We, and others, have previously reported such viral vectored vaccines to be safe and immunogenic for T cells and antibodies in healthy adult UK and US volunteers when delivering numerous P. falciparum antigens, including the pre-erythrocytic antigen multiple-epitope string fused to thrombospondinrelated adhesion protein (ME-TRAP) (35) and circumsporozoite protein (PfCSP) (36), as well as the blood-stage antigens merozoite surface protein 1 (PfMSP1) (37) and apical membrane antigen 1 (PfAMA1) (38,39). In 2014 and 2015, the same adenovirus-poxvirus vectored vaccine technologies were developed rapidly for Ebola (40).
Here, we report the safety and immunogenicity of a similar approach in an open-label dose-escalation phase Ia study in healthy UK adults using replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) encoding PvDBP_RII from the Salvador I (SalI) reference strain of P. vivax. These vaccines have been previously shown to be immunogenic in mice and rabbits (27). Now we show that these vaccines demonstrate a favorable safety profile in malarianaive adults, and confirm to our knowledge for the first time that substantial PvDBP_RII-specific antibodies and B cell and T cell responses can be induced by immunization in humans. Vaccine-induced serum antibodies were capable of inhibiting the in vitro binding of vaccine homologous and heterologous variants of recombinant PvDBP_RII to the DARC receptor.

Twenty-four healthy adult volunteers were enrolled into the VAC051 trial to test the ChAd63-MVA PvDBP_RII vaccine
in an open-label, dose-escalation study design. Thirty UK adult volunteers were screened in total, of which 24 were enrolled ( Figure 1). Four volunteers were recruited to groups 1 and 2A, and 8 volunteers to groups 2B and 2C. In total, 15 females and 9 males were enrolled. The mean age of volunteers was 25 years 9 months (range 18-40 years). Four volunteers were enrolled into group 1 and received 5 × 10 9 viral particles (vp) of the ChAd63 PvDBP_RII vaccine. Following a safety review, the dose of ChAd63 PvDBP_RII was increased to 5 × 10 10 vp for group 2. Four volunteers in group 2A received ChAd63 PvDBP_RII alone, while volunteers in groups 2B and 2C received ChAd63 PvDBP_RII followed 8 weeks later with a boost vaccination of MVA PvDBP_RII at a dose of 1 × 10 8 plaque-forming units (PFU) or 2 × 10 8 PFU, respectively. One volunteer withdrew from group 2B prior to the MVA PvDBP_RII vaccination due to personal commitments and was not replaced, resulting in 23 volunteers completing follow-up as per protocol.
ChAd63 and MVA PvDBP_RII show a favorable safety profile in healthy UK adult volunteers. There were no serious adverse events (AEs) or unexpected reactions during the course of the trial and no volunteers withdrew due to vaccine-related AEs. ChAd63 PvDBP_RII and MVA PvDBP_RII demonstrated favorable safety profiles, similar to those seen in previous clinical trials with the same viral vectors recombinant for P. falciparum malaria antigens (35)(36)(37)(38). All AEs following ChAd63 PvDBP_RII 5 × 10 9 vp were mild, as were the vast majority in group 2, although some volunteers did report moderate or severe AEs following immunization with the full dose. The higher dose of MVA PvDBP_RII was more reactogenic than the lower dose, with half of the volunteers reporting at least 1 severe AE, although no systemic AE was reported as severe for more than 24 hours. The maximum severities of solicited local and systemic AEs reported by volunteers following each vaccination are shown in Figure 2. All unsolicited AEs considered possibly, probably, or definitely related to either vaccination were mild in nature (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.93683DS1). There was only 1 laboratory AE following ChAd63 PvDBP_RII that was considered possibly, probably, or definitely related to vaccination: a mild lymphopenia in 1 volunteer vaccinated with 5 × 10 10 vp. Similarly, there was only 1 laboratory AE following MVA PvDBP_RII that was considered possibly, probably, or definitely related to vaccination: a moderate eosinophilia in 1 volunteer vaccinated with 1 × 10 8 PFU, which peaked more than 4 weeks after vaccination. Both of these laboratory AEs resolved spontaneously.
ChAd63 and MVA PvDBP_RII expand IFN-γ T cell responses in healthy UK adult volunteers. The kinetics and magnitude of the PvDBP-specific T cell response were assessed over time by ex vivo IFN-γ ELISPOT following restimulation of peripheral blood mononuclear cells (PBMCs) with 20-mer peptides overlapping by 10 amino acids (aa) spanning the entire PvDBP_RII insert present in the vaccines (Figure 3). Vaccination with ChAd63-MVA PvDBP_RII induced antigen-specific T cell responses in all volunteers, with individual responses shown in Supplemental Figure 1 and median responses to the total vaccine insert shown for each group in Figure 3A. Following ChAd63 PvDBP_RII prime, there was no significant difference between median responses in the lower-dose group 1 in comparison with group 2 at the peak of the response on  detected in all 6 of the peptide pools used in the ELISPOT assay (Supplemental Figure 2). Following the peak at day 63, responses contracted but were maintained above baseline at the end of the study period, with significantly better maintained responses at day 140 in group 2C as compared with group 2B (median 1,871 vs. 385 SFU/million PBMCs) (P = 0.03, Mann-Whitney test) ( Figure 3D).

ChAd63 and MVA PvDBP_RII induce serum antibody responses and memory B cells in healthy UK adult volunteers.
The kinetics and magnitude of the anti-PvDBP_RII serum IgG antibody response were assessed over time by ELISA against recombinant protein ( Figure 4). Priming vaccination with 5 × 10 10 vp ChAd63 PvDBP_RII followed by MVA PvDBP_RII boost induced antigen-specific IgG responses in all volunteers (groups 2B and 2C), with individual responses shown in Supplemental Figure 3 and median responses shown for each group in Figure 4A. Responses are reported in μg/ml following conversion of ELISA arbitrary units (AU) by calibration-free concentration analysis (CFCA) (Supplemental Figure 4). Following ChAd63 PvDBP_RII prime with 5 × 10 9 vp, none of the 4 volunteers showed a detectable response on day 28, in contrast to 12 of 20 volunteers who did show a response (median 0.3, range 0-2.3 μg/ml, n = 20) following priming with 5 × 10 10 vp (P = 0.07, Mann-Whitney test) ( Figure 4B). Responses were subsequently maintained in group 2 volunteers prior to administration of MVA PvDBP_RII, which led to a boost as measured 4 weeks later on day 84 ( Figure 4A) -this reached significance for group 2C versus 2A (Kruskal-Wallis test with Dunn's multiple comparison test) ( Figure 4C). Responses in group 2C (median 15.6, range 10.5-27.2 μg/ml, n = 8) were also modestly, but significantly, higher than in group 2B (median 8.8, range 5.5-23.7 μg/ml, n = 8) at this peak time point (P = 0.014, Mann-Whitney test). Serum antibody responses decreased by day 140 but were well maintained above preboost levels, with no significant difference between groups 2B and 2C (P = 0.34, Mann-Whitney test) ( Figure 4D). Day 84 plasma were also tested against a panel of overlapping 20-mer linear peptides; however, few responses were detected above background, suggesting the vast majority of vaccine-induced anti-PvDBP_RII IgG recognize conformational, as opposed to linear, epitopes (Supplemental Figure 5).
The serum antibody response against PvDBP_RII as measured by ELISA at day 84 was composed of IgG1 and modest levels of IgG3 ( Figure 4E), with little to no IgG2, IgG4, IgA, or IgM detectable above baseline (day 0) levels (Supplemental Figure 6). The avidity of the anti-PvDBP_RII IgG, as measured by a NaSCN-displacement ELISA, was similar at day 84 for all volunteers in groups 2B and 2C, with the IC 50 ranging from 1.9 to 4.1 M. Avidity could only be measured for 1 vaccinee in group 2A at this time point with an IC 50 of 2.8 M, suggesting no change following MVA PvDBP_RII boost ( Figure 4F).
Previous studies have shown that antibody-secreting cells (ASCs) can be detected in peripheral blood for a short time (around day 7) after MVA boost when using the ChAd63-MVA regimen (41,42). PvDBP_ RII-specific ASC responses were assessed by ex-vivo ELISPOT using frozen PBMCs collected at the day 63 visit for volunteers in groups 2B and 2C. Median responses of 49 versus 159 ASCs per million PBMCs were observed, respectively, but there was no significant difference between the 2 groups (P = 0.69, Mann-Whitney test) ( Figure 5A). ASC responses across both groups showed a trend to associate with peak serum antibody responses at day 84, but this did not reach significance ( Figure 5B).
Memory B cell (mBC) responses were also measured using an established cultured ELISPOT protocol, whereby mBCs within PBMCs undergo a 6-day polyclonal stimulation to form ASCs, which are then Responses are reported as number of mBC-derived PvDBP_RII-specific ASCs per million cultured PBMCs ( Figure 5C), and as a percentage of total IgG-secreting ASCs ( Figure 5D); in both cases these were significantly higher in group 2C than 2B (Mann-Whitney test). These mBC responses across both groups also significantly correlated with peak serum antibody responses at day 84 ( Figure 5, E and F).

C L I N I C A L M E D I C I N E
Vaccine-induced antibodies inhibit PvDBP_RII-DARC binding in vitro. We next assessed the ability of vaccineinduced serum IgG to inhibit binding of recombinant vaccine-homologous PvDBP_RII (SalI) to its receptor (in this case the recombinant N-terminal region of DARC), using an in vitro ELISA methodology in Oxford. Day 84 sera were tested using a 2-fold dilution series starting at 1:5 and through to 1:640, with percentage binding inhibition calculated for each volunteer using their matched day 0 serum sample as the baseline control. Example binding-inhibition curves are shown (Supplemental Figure 7A), and 50% binding-inhibition titers were interpolated from these data ( Figure 6A). One sample in group 2A showed a weak 50% binding-inhibition titer of 1:16. All samples from groups 2B and 2C showed binding inhibition with median 50% titers of 1:137 (range 1:14-1:248) and 1:168 (range 1:52-1:352), respectively. To further assess the quality of the vaccineinduced antibody response, these titers were used to calculate the concentration of anti-PvDBP_RII polyclonal IgG that gives 50% binding inhibition in each individual ( Figure 6B). Across all groups, the median levels were comparable, requiring 128, 96, and 105 ng/ml in groups 2A, 2B, and 2C, respectively. However, there was over a 10-fold range across all 16 individuals, with the best responder (in group 2C) only requiring 39 ng/ml, versus the worst responder requiring 540 ng/ml (in group 2B). These data suggest that interindividual qualitative differences exist in terms of the binding-inhibitory capacity of the polyclonal vaccine-induced IgG response.
Given that naturally acquired binding-inhibitory anti-PvDBP_RII antibodies can be strain specific (43,44), we next proceeded to test the day 0 and 84 sera from group 2 against an established panel of recombinant PvDBP_RII alleles (Table 1) using methodology developed at ICGEB, India ( Figure 6, C-F). No binding inhibition was observed for any of the day 0 samples against any PvDBP_RII variant. Data for the SalI variant showed very similar results to those observed with the Oxford assay (Spearman's correlation r s = 0.67, P = 0.002, n = 19). Day 84 sera also showed similar 50% binding-inhibition profiles for the 3 other variants of PvDBP_RII (PvAH, PvO, and PvP), with the same sample positive in group 2A, and median 50% binding-inhibition titers greater than 1:100 for both groups 2B and 2C for all test variants. At the individual level, all samples showed binding inhibition against each variant of PvDBP_RII, but the 50% binding-inhibition titers were variable, again consistent with qualitative differences in each polyclonal response (Supplemental Figure 7B). Interestingly, the individual titers were frequently highest against the vaccine-heterologous PvAH or PvO alleles.
Finally we tested the day 84 sera against an allele of PvDBP_RII present in the HMP013 Indian strain of P. vivax, which has recently been cryobanked for use as an inoculum in blood-stage CHMI clinical trials (16). After generating a draft assembly of HMP013 (see supplementary material), analysis of the PvDBP_ RII sequence from this strain (Table 1) revealed 10 polymorphic positions, of which 5 were not shared with the other variants tested in this study, including some in subdomain 2 (SD2) close to the site shown to bind to aa 19-30 of DARC ( Figure 7A). Recombinant PvDBP_RII (HMP013) was subsequently generated and used in the Oxford assay. Binding-inhibition curves were similar to those previously observed with the SalI allele ( Figure 7B). Fifty percent binding-inhibition titers were interpolated ( Figure 7C), with the data for group 2 again showing a similar profile to those observed with the SalI variant ( Figure 7D).

Discussion
This phase Ia dose-escalation and safety study reports the first data in humans for a vaccine targeting the PvDBP_RII antigen from the blood-stage P. vivax malaria parasite. We have shown in healthy malaria-naive The Salvador I (SalI) reference sequence is shown in bold. Amino acid polymorphisms are indicated for the region II of the P. vivax Duffy-binding protein (PvDBP_RII) variants (P, O, and AH) described previously (60), plus the P. vivax HMP013 strain. Amino acids that are the same as the reference sequence are indicated by a period, and a hyphen indicates an insertion/deletion. A HMP013 has a leucine insertion between V429 and P430 in the SalI reference sequence. adult volunteers that a recombinant ChAd63-MVA heterologous prime-boost immunization regimen can induce binding-inhibitory antigen-specific serum antibody responses in addition to B and T cell responses. ChAd63 and MVA recombinant for PvDBP_RII also demonstrated a favorable safety profile. Reactogenicity of the ChAd63 PvDBP_RII vector was similar to that seen consistently with the same doses of ChAd63 vectored vaccines encoding the P. falciparum pre-erythrocytic malaria antigens ME-TRAP or PfCSP (35,36) and the blood-stage antigens PfMSP1 or PfAMA1 (37,38,42,45). In more recent years, safety and immunogenicity data for the ME-TRAP vaccines have been reported in adults, children, and infants residing in malaria-endemic areas (46). Our data with ChAd63 PvDBP_RII add to the growing body of evidence that this simian adenovirus vector is safe for clinical use. Reactogenicity of the MVA PvDBP_RII vector appeared to be more pronounced than the ChAd63 vector and increased with dose, again consistent with previous experience of using this orthopoxvirus vector for P. falciparum vaccines (37,38) as well as other disease targets such as respiratory syncytial virus (47), hepatitis C virus (48), Ebola virus (40), HIV, and Mycobacterium tuberculosis (49). Indeed, the clinical safety of MVA as a recombinant vaccine vector for many infectious diseases and cancer is now well documented. The ChAd63-MVA delivery platform was originally developed to induce T cell responses against a target blood-stage malaria antigen in humans (32,33). Similar to our data in mice using this vaccine (27), the results presented here show that PvDBP_RII-specific IFN-γ T cell responses were induced and peaked at median levels of greater than 2,000 SFU/million PBMCs following the MVA boost. The kinetics and magnitude of this response are extremely similar to those previously seen with the same vectors encoding P. falciparum antigens (35)(36)(37)(38). These previous studies using the ChAd63-MVA regimen, as well as other studies using alternative ChAd serotypes followed by MVA boost (40,47,48), have routinely shown that a mixed antigen-specific CD4 + /CD8 + T cell response is induced in humans.
The ELISPOT data showed that the IFN-γ T cell responses were spread across all 6 peptide pools spanning the PvDBP_RII antigen. A previous study assessed IFN-γ and IL-10 T cell responses to aa 291-460 of PvDBP_RII in naturally exposed children and adults from Papua New Guinea. These data showed that age-dependent low-level responses are detectable in a subset of individuals following natural P. vivax infection (<150 SFU/million PBMCs using a 3-day cultured ELISPOT protocol, as opposed to the overnight stimulation used in the ex vivo assay reported here) (50). Five PvDBP_RII T cell epitopes were identified by peptide mapping, with 3 of these containing polymorphic residues leading to variant-specific cellular responses (50). Nevertheless, the contribution of T cell responses to blood-stage immunity against P. vivax remains unclear. In the case of P. falciparum, clinical trials using whole-parasite immunization (51) or ChAd63-MVA vectors encoding PfMSP1 or PfAMA1 (45) failed to show an impact on blood-stage parasite growth following CHMI despite strong T cell induction by vaccination. However, recent data from other CHMI studies show that, unlike P. falciparum, blood-stage P. vivax activates cytotoxic CD38 + CD8 + T cells that could target parasites residing within MHC class I-expressing reticulocytes (52), suggesting that it may be possible for effector T cells to play a more direct role against this species of human malaria parasite.
In agreement with preclinical data in mice and rabbits (27), the ChAd63-MVA prime-boost regimen also induced PvDBP_RII-specific serum IgG antibody responses, peaking at a median of 0.3 μg/ml after ChAd63 prime and 15.6 μg/ml after MVA boost in the full-dose vaccination groups. The kinetics and magnitude of the antigen-specific IgG, ASC, and mBC responses induced here in malaria-naive humans are consistent with those reported for the same vectors encoding the P. falciparum blood-stage antigens PfMSP1 and PfAMA1 (37,38,41,42). With regard to the PvDBP_RII-specific antibody concentrations, these were lower than those seen following ChAd63-MVA immunization with PfAMA1 (37,42) and PfMSP1 (37), but 8-fold higher than with PfCSP (36). Similar to these P. falciparum vaccines (42,53), the anti-PvDBP_ RII serum IgG response was largely composed of IgG1 and some IgG3, with moderate avidity as measured by NaSCN-displacement ELISA. These qualitative aspects of the vaccine-induced antibody responses are consistent with those observed to the same antigen following natural infection in endemic populations (54,55); however, the contributions of antibody isotype, affinity, and avidity to protection against the P. vivax merozoite remain poorly understood.
Studies of naturally acquired immunity following P. vivax exposure have reported the induction of strain-specific immunity (43,44) and numerous sequence polymorphisms, consistent with immune evasion, have been found within the PvDBP_RII antigen, with the majority localized to SD2 (24,56). Nevertheless, high-titer naturally acquired BIAbs that block binding of diverse PvDBP_RII alleles from P. vivax field isolates have also been reported, albeit at low frequency (30,31). Once acquired, these antibodies are maintained and associate with clinical immunity to P. vivax. In contrast to these epidemiological data, preclinical immunogenicity studies with the SalI allele of PvDBP_RII have shown that this immunogen is capable of eliciting high-titer, cross-reactive BIAbs, as assessed using the ELISA-based binding-inhibition assay (28). Consistent with these data, our studies here suggest that PvDBP_RII vaccination of humans can elicit antibodies that qualitatively differ from those induced by natural exposure. Across all vaccinees who received the ChAd63-MVA regimen, anti-PvDBP_RII responses were induced that blocked binding of variant PvDBP_RII alleles to DARC in vitro including one from the HMP013 strain, suggesting this strain would be suitable to test vaccine efficacy in a future phase IIa CHMI study (16). Encouragingly, median 50% binding-inhibition titers greater than 1:100 were consistently observed for all test variants; these are higher than the ~1:20 titers reported by others using a similar assay for naturally acquired, strain-transcending BIAbs in a limited number of children in Papua New Guinea and associated with clinical immunity (30). However, this association with clinical immunity has never been formally demonstrated in the context of a vaccine clinical trial. ChAd63-MVA PvDBP_RII is the first candidate vaccine against blood-stage P. vivax to reach clinical testing. It is therefore vital in a future CHMI efficacy study to assess whether vaccineinduced BIAbs associate with control of blood-stage parasite growth.
The data obtained from this study also suggested interindividual qualitative differences in terms of the polyclonal anti-PvDBP_RII IgG response, as would be anticipated following human vaccination. A recent cohort study in the Brazilian Amazon has suggested that genetic variation in HLA class II genes can influence antibody responses against PvDBP_RII following natural P. vivax infection (57). Similarly, studies of naturally acquired anti-PvDBP_RII IgG responses (58) as well as mouse monoclonal antibodies (59, 60) have reported linear and conformational epitopes. Here we failed to detect linear responses by ELISA using a peptide array, and our ongoing work will focus on elucidating epitopes recognized by vaccine-induced human B cells in order to guide future immuno-monitoring. Further ongoing work is seeking to assess antibody function against P. vivax parasites. Importantly, we have previously reported that adenovirus-MVA immunization of mice and rabbits elicits antibodies that recognize native parasite antigen by immunofluorescence assay (IFA) (27). Future studies will focus on optimizing short-term invasion-inhibition assay methodology (61) to allow for functional testing of vaccine-induced antibodies from human volunteers in clinical trials.
Overall, the association between Duffy negativity and protection against blood-stage P. vivax infection was first reported in 1976 (15), but until now this observation has not been translated into a clinical vaccine candidate. The intervening years have seen the PvDBP_RII-DARC interaction described in molecular detail and related immuno-epidemiology extensively studied in the field. Here we extend this work and demonstrate, possibly for the first time, that substantial PvDBP_RII-specific antibodies as well as B cell and T cell responses can be induced safely by immunization in humans, using a leading viral vectored delivery strategy that is in clinical development for numerous difficult and emerging diseases and cancer. Encouragingly for the P. vivax vaccine field, a second PvDBP_RII protein-based vaccine formulated in the emulsified version of glucopyranosyl lipid adjuvant (GLA-SE) has also recently entered a phase I clinical trial in India (CTRI/2016/09/007289). The demonstration in parallel of a blood-stage CHMI model for vaccine testing using P. falciparum (62), and the banking of similar blood-stage inocula for P. vivax (16), should allow for this ChAd63-MVA vaccine and others to progress to rapid phase IIa proof-of-concept efficacy testing in the near future.

Methods
Detailed methods are provided in supplemental methods.
ChAd63 and MVA PvDBP_RII vaccines. The design, production, and preclinical testing of the viral vector vaccines have been reported previously (27). Briefly, both recombinant viruses express the same 984-bp coding sequence of PvDBP_RII from the SalI strain of P. vivax, aa D194-T521 (GenBank Accession DQ156512). ChAd63 PvDBP_RII was manufactured under current Good Manufacturing Practice (cGMP) conditions by the Clinical Biomanufacturing Facility (CBF), University of Oxford, UK, and MVA PvDBP_RII was manufactured under cGMP conditions by IDT Biologika GmbH, Germany, both as previously described (37).
Study design and approvals. The VAC051 study was a phase Ia open-label, dose-escalation, first-inhuman, nonrandomized trial of the viral vectored vaccines ChAd63 PvDBP_RII and MVA PvDBP_RII given in a prime-boost regimen with an 8-week interval. The study was conducted at the Centre for Clinical Vaccinology and Tropical Medicine (CCVTM), University of Oxford, Oxford, UK. The study received ethical approval from the Oxfordshire Research Ethics Committee A in Oxford, UK (REC reference 13/ SC/0001). The study was also reviewed and approved by the UK Medicines and Healthcare products Regulatory Agency (MHRA, reference 21584/0312/001-0001). Volunteers signed written consent forms and consent was verified before each vaccination. The trial was registered on Clinicaltrials.gov (NCT01816113) and was conducted according to the principles of the current revision of the Declaration of Helsinki 2008 and in full conformity with the ICH guidelines for Good Clinical Practice (GCP). The primary endpoint of the study was to assess the safety of ChAd63 PvDBP_RII and MVA PvDBP_RII, with a secondary endpoint to assess immunogenicity.