Introduction The development of a highly effective and deployable malaria vaccine remains an urgent priority for improving global public health. Despite recent strides in disease prevention and control, the Plasmodium falciparum human malaria parasite continues to exert a huge toll in terms of morbidity and mortality (Murray et al., 2012). The most advanced malaria subunit vaccine, a virus-like particle known as RTS,S, has shown only modest efficacy in young children in Phase III clinical trials (Agnandji et al., 2012), and thus new approaches are urgently needed (Moorthy et al., 2013). RTS,S induces antibodies that reduce liver infection by the parasite (Foquet et al., 2014). An alternative and complementary strategy is to vaccinate against the subsequent blood-stage infection (which causes clinical disease and against which natural immunity is slowly acquired). Such a vaccine could prevent death and reduce incidence of disease, parasitemia, and onward transmission (Hill, 2011). However, despite 25 years of development, vaccine candidates targeting P. falciparum’s asexual blood stage have failed to overcome the challenge posed by the parasite’s antigenic diversity. Two of the most critical road blocks have included exceptionally high thresholds for protective levels of antibody against known target antigens, coupled with problematic levels of antigen polymorphism. To date, no vaccine candidate has overcome these hurdles to achieve in vivo protection in human clinical trials (Goodman and Draper, 2010; Thera et al., 2011). In previous nonhuman primate (NHP) studies (which provide the only opportunity to study the effect of vaccines against an uninterrupted P. falciparum blood-stage infection), blood-stage vaccine candidates have proven protective only against vaccine-homologous parasite lines, and only when administered with non-human-compatible adjuvants (Dutta et al., 2009; Lyon et al., 2008). P. falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a recently identified merozoite protein, secreted from the apical organelles of the parasite during the red blood cell (RBC) invasion process (Baum et al., 2009). In vitro data have identified PfRH5 as the highest priority target in the blood-stage malaria vaccine field for over a decade (Douglas et al., 2011). Antibodies induced by PfRH5 vaccination of mice and rabbits overcome the two major difficulties outlined above: (i) antibodies can block erythrocyte invasion to high efficiency (with lower EC50 in terms of μg/ml antigen-specific antibody than against all other known antigens) (Douglas et al., 2014; Miura et al., 2009; Williams et al., 2012) and (ii) most importantly, these antibodies cross-inhibit all P. falciparum lines and field isolates tested to date (Bustamante et al., 2013; Douglas et al., 2011; Reddy et al., 2014; Williams et al., 2012). The PfRH5 protein is now known to mediate a critical nonredundant interaction with the human RBC surface protein basigin during invasion (Crosnier et al., 2011). The PfRH5 gene is also refractory to genetic deletion (Baum et al., 2009; Hayton et al., 2008), unlike many other blood-stage antigens, confirming the essential nature of its function. In the context of natural infection, PfRH5 does not appear to be a dominant target of naturally acquired immune responses in endemic populations (Douglas et al., 2011; Tran et al., 2014; Villasis et al., 2012), but when detected, such antibody responses correlate with protective clinical outcome (Tran et al., 2014), and affinity-purified anti-PfRH5 human antibodies can neutralize parasites in vitro (Patel et al., 2013; Tran et al., 2014). The high degree of PfRH5 sequence conservation is thus associated with low-level natural immune pressure, but also functional constraints linked to basigin binding. Importantly, it has been shown that minimal amino acid substitutions in PfRH5 account for loss of basigin binding and/or host RBC tropism (linked to binding basigin orthologs from other species), suggesting the antigen may not easily escape vaccine-induced immune pressure (Hayton et al., 2008, 2013; Wanaguru et al., 2013). However, to date, no study has assessed the protective efficacy of PfRH5-based vaccines in vivo, and it remains unclear whether the encouraging observations made in vitro using an assay of parasite neutralization will translate into biologically relevant antiparasitic activity. This question is of particular importance, given the current lack of a clear correlate of vaccine efficacy against blood-stage infection in humans (Duncan et al., 2012) and the need to design improved strain-transcending malaria vaccines that can be progressed to clinical development. In this study, we quantitatively assessed the immunogenicity of PfRH5-based vaccines delivered to Aotus monkeys by three different immunization regimens, including protein-in-adjuvant formulations (de Cassan et al., 2011) and an adenovirus/poxvirus vectored platform previously optimized for Phase I/IIa clinical development (Draper et al., 2008; Sheehy et al., 2012). We also evaluated the protective efficacy of these vaccines against a stringent vaccine-heterologous P. falciparum challenge (Stowers and Miller, 2001). This study enabled us to monitor the ability of PfRH5-based vaccines to both control and clear a virulent blood-stage infection. We report that significant protection against challenge with heterologous-strain blood-stage P. falciparum can be achieved in vivo by these vaccines, including when using the human-compatible viral vectored delivery platform. This protection was associated with anti-PfRH5 antibody concentration and parasite-neutralizing activity, supporting the use of this assay to predict the in vivo efficacy of future vaccine candidates. These results suggest that PfRH5-based vaccines have the potential to achieve strain-transcending efficacy in humans. Results Evaluation of PfRH5 Vaccine Efficacy in Aotus Monkeys 31 Aotus nancymaae monkeys were randomized to groups that received protein-in-adjuvant and/or viral vectored vaccination regimes targeting either P. falciparum RH5 or apical membrane antigen 1 (PfAMA1), a well-studied comparator antigen that elicits strain-specific antibodies (Dutta et al., 2009; Remarque et al., 2008; Thera et al., 2011) (Figure 1A). The PfRH5 protein immunogen was pure (Figure S1A) and shown to be correctly folded by demonstration of binding to its receptor, basigin (Crosnier et al., 2011) (Figure S1B). Group A received sham vaccines, chimpanzee adenovirus serotype 63 (ChAd63) expressing Renilla luciferase (RLuc) prime, PBS with Abisco-100 adjuvant boost; Group B received PfRH5 protein with complete or incomplete Freund’s adjuvant (CFA, IFA); Group C received ChAd63 expressing PfRH5 prime, PfRH5 protein with Abisco-100 boost; Group D received ChAd63-PfRH5 prime, modified vaccinia virus Ankara (MVA) expressing PfRH5 boost; and Group E received ChAd63-PfAMA1 prime, PfAMA1 protein with Abisco-100 boost. The ChAd63-MVA vaccine delivery platform used here has now been progressed to human clinical testing for a wide variety of difficult disease targets, including malaria, HIV-1, and hepatitis C virus (de Cassan and Draper, 2013; Draper and Heeney, 2010), while the use of mixed-modality adenoviral priming-protein-boost regimens has shown promise in small animals as well as initial clinical studies (de Cassan et al., 2011; Draper et al., 2010) (Hodgson et al., 2014). In the case of this study, the PfRH5 vaccines encoded the 3D7 allele of the antigen, while for PfAMA1 the ChAd63 vector expressed two alleles of the antigen (3D7 and FVO), and FVO allele PfAMA1 protein was used for the boost. The Group A sham-vaccinated animals served as protocol-specified infectivity controls in order to confirm consistent infection by the FVO parasite inoculum and its appropriate adaptation to growth in Aotus. To evaluate the protective efficacy of the vaccines, animals were challenged 15 days after the final vaccination by intravenous administration of 104 PfRH5-vaccine-heterologous FVO strain P. falciparum infected red blood cells (iRBC) taken from a donor monkey. The parasitemia (Figures 1B–1F) and hematocrit (Hct) (Figures S1C–S1G) in the challenged animals were monitored over time. Challenge infection with this parasite line has proven highly virulent in Aotus nancymaae over the course of numerous studies, requiring treatment in all control animals administered complete Freund’s adjuvant without a blood-stage vaccine antigen (n = 55, Table S1 and Supplemental Information). In contrast, none of the animals immunized here with PfRH5 protein in Freund’s adjuvant (Group B) required treatment. Efficacy in this group was significant, both comparing treatment status versus adjuvant-matched historical controls (the protocol-specified primary analysis for this group; Kendall’s τB = 0.703, p 50% GIA at 2.5 mg/ml, total IgG GIA EC50 was calculated in terms of total IgG concentration in the well by linear interpolation. The total IgG concentration in each plasma sample was measured using Protein A biosensors on a Fortebio Blitz instrument (ForteBio). For each animal achieving >50% GIA at 2.5 mg/ml, the GIA50 titer was then calculated by dividing the plasma total IgG concentration by the total IgG GIA EC50. Analyses and Statistics Throughout, all reported p values are for two-tailed tests. Vaccine efficacy endpoints were recorded, as used in a previous Aotus-P. falciparum challenge study (Lyon et al., 2008) and a study of P. knowlesi infection of rhesus macaques (Mahdi Abdel Hamid et al., 2011). Kendall’s tau-b was used to test a null hypothesis of equivalent outcome between Group B and historical Freund’s control animals (see Table S1 and Supplemental Information) using the ordinally ranked outcome data. As a secondary efficacy outcome measure for this group (using non-adjuvant-matched control data from the current study), LCP was compared between Groups B and A by Mann-Whitney test. The protocol-specified primary analysis of efficacy in Groups C, D, and E was comparison of LCP in each group to Group A by Mann-Whitney test with Bonferroni correction for multiple comparison. A post hoc secondary analysis of efficacy in terms of effect upon time to treatment was performed using a Mann-Whitney test with Bonferroni correction for multiple comparison, comparing each of Groups B, C, D, and E to Group A. The majority of immunological parameters were nonnormally distributed, and thus, unless detailed otherwise in the Supplemental Information, analyses of association between immunological parameters and continuous outcome variables were performed by Spearman’s rank correlation. The protocol-specified primary analysis for a correlate of protection, in the event that GIA EC50 data could not be estimated for every animal (as was the case here for a number of the animals in Groups C and E), was examination of the correlation between GIA at a fixed total IgG concentration and IVIG. Author Contributions A.D.D., G.C.B., K.M., C.A.L., K.A.E., Y.W., G.J.W., A.G.L., and S.J.D. designed and reviewed the study and interpreted the data; A.D.D., G.C.B., J.A.V., and A.J.S. performed the cellular immunogenicity assays; A.D.D., A.D., K.M., K.H.L., K.H.M., K.A.H., C.A.L., and S.J.D. performed the humoral immunogenicity assays; A.D.D., C.C., S.J.B., J.J.I., D.G.W.A., A.V.T., Y.W., G.J.W., and S.J.D. prepared the proteins and various vaccine constructs; A.D.D., G.C.B., C.M.L., L.E.L., J.A.V., K.P.L., and Y.W. assisted with the malaria challenge and parasitological monitoring; L.A.L.-R. and J.T.M. undertook the clinical care of the Aotus monkeys; A.D.D. and S.J.D. performed the data and statistical analyses; and A.D.D. and S.J.D. led the study and wrote the paper with all the co-authors.