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      Kinetics of antibody responses to PfRH5-complex antigens in Ghanaian children with Plasmodium falciparum malaria

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          Abstract

          Plasmodium falciparum PfRH5 protein binds Ripr, CyRPA and Pf113 to form a complex that is essential for merozoite invasion of erythrocytes. The inter-genomic conservation of the PfRH5 complex proteins makes them attractive blood stage vaccine candidates. However, little is known about how antibodies to PfRH5, CyRPA and Pf113 are acquired and maintained in naturally exposed populations, and the role of PfRH5 complex proteins in naturally acquired immunity. To provide such data, we studied 206 Ghanaian children between the ages of 1–12 years, who were symptomatic, asymptomatic or aparasitemic and healthy. Plasma levels of antigen-specific IgG and IgG subclasses were measured by ELISA at several time points during acute disease and convalescence. On the day of admission with acute P. falciparum malaria, the prevalence of antibodies to PfRH5-complex proteins was low compared to other merozoite antigens (EBA175, GLURP-R0 and GLURP-R2). At convalescence, the levels of RH5-complex-specific IgG were reduced, with the decay of PfRH5-specific IgG being slower than the decay of IgG specific for CyRPA and Pf113. No correlation between IgG levels and protection against P. falciparum malaria was observed for any of the PfRH5 complex proteins. From this we conclude that specific IgG was induced against proteins from the PfRH5-complex during acute P. falciparum malaria, but the prevalence was low and the IgG levels decayed rapidly after treatment. These data indicate that the levels of IgG specific for PfRH5-complex proteins in natural infections in Ghanaian children were markers of recent exposure only.

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          Reticulocyte-binding protein homologue 5 - an essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum.

          Invasion of erythrocytes is a prerequisite in the life history of the malaria parasite. Members of the reticulocyte-binding homologue family (PfRh) have been implicated in the invasion process and in some cases have been shown to act as adhesins, binding to specific receptors on the erythrocyte surface. We have identified a further, putatively essential, PfRh family member in the most virulent human malaria Plasmodium falciparum, called PfRh5, which binds to an unknown class of glycosylated receptors on the erythrocyte surface. This protein is an atypical PfRh family member, being much smaller than others and lacking a transmembrane and cytosolic region at the C-terminus. This suggests it may be part of a functional protein complex. PfRh5 localises to the rhoptries in merozoites and follows the tight junction during the process of erythrocyte invasion. Furthermore, rabbit immune serum raised against a portion of the ecto-domain, inhibits parasite invasion in vitro. We hypothesise an essential role for the PfRh5 adhesin in erythrocyte selection and commitment to invasion. Given its small size, we believe PfRh5 may prove to be a valuable candidate for inclusion in a multi-component anti-malarial vaccine.
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            A PfRH5-Based Vaccine Is Efficacious against Heterologous Strain Blood-Stage Plasmodium falciparum Infection in Aotus Monkeys

            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.
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              Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5.

              There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5-based malaria prevention efforts.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: ResourcesRole: Validation
                Role: InvestigationRole: Resources
                Role: Formal analysisRole: Methodology
                Role: Formal analysisRole: Funding acquisitionRole: MethodologyRole: ResourcesRole: Supervision
                Role: Funding acquisitionRole: ResourcesRole: Supervision
                Role: Funding acquisitionRole: ResourcesRole: Supervision
                Role: Data curationRole: Funding acquisitionRole: ResourcesRole: Supervision
                Role: Funding acquisitionRole: InvestigationRole: ResourcesRole: Supervision
                Role: Data curationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                8 June 2018
                2018
                : 13
                : 6
                : e0198371
                Affiliations
                [1 ] Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
                [2 ] Centre for Medical Parasitology at Department of Immunology and Microbiology (ISIM), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
                [3 ] Department of Infectious Diseases Copenhagen, University Hospital (Rigshospitalet), Copenhagen, Denmark
                [4 ] Department of Clinical Microbiology, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark
                [5 ] The Jenner Institute, University of Oxford, Oxford, United Kingdom
                [6 ] West Africa Centre for Medical Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Legon, Ghana
                [7 ] Hohoe Municipal Hospital, Hohoe, Ghana
                Universidade Federal de Minas Gerais, BRAZIL
                Author notes

                Competing Interests: The Novo Nordisk Foundation is not a commercial source and had no influence on this manuscript or anything relating to employment, consultancy, patents, products in development, marketed products, etc. Therefore, this does not alter our adherence to PLOS ONE policies on sharing data and materials.

                [¤]

                Current address: Ketu South Municipal Hospital, Aflao, Ghana

                Author information
                http://orcid.org/0000-0002-1140-5527
                http://orcid.org/0000-0001-6510-495X
                Article
                PONE-D-18-03997
                10.1371/journal.pone.0198371
                5993283
                29883485
                15dcd8b3-d2c9-4bb6-8054-96b4f3d035cc
                © 2018 Partey et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 6 February 2018
                : 17 May 2018
                Page count
                Figures: 4, Tables: 2, Pages: 14
                Funding
                Funded by: Consultative Committee for Development Research
                Award ID: DFC 12 081RH
                Award Recipient :
                Funded by: Welcome Trust
                Award ID: 106917/Z/15/Z
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004191, Novo Nordisk;
                Award ID: NNF170C0026778
                Award Recipient :
                The study was mainly supported by The Consultative Committee for Development Research [grant number DFC 12 081RH] http://dfcentre.com. Other funding resources: Wellcome Trust [grant number 106917/Z/15/Z] https://wellcome.ac.uk/ and Novo Nordisk Foundation [grant number NNF170C0026778] http://novonordiskfonden.dk. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                Medicine and Health Sciences
                Parasitic Diseases
                Malaria
                Medicine and Health Sciences
                Tropical Diseases
                Malaria
                Biology and Life Sciences
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                Immune Physiology
                Antibodies
                Medicine and Health Sciences
                Physiology
                Immune Physiology
                Antibodies
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                Immunology
                Immune System Proteins
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                Immunology
                Immune System Proteins
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                Parasitology
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                Research and Analysis Methods
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