Kinetic constraints around the development of a malaria vaccine. activity, but absence of blockade does not predict absence of functional activity; ii) neutralizing mAbs bind spatially-related epitopes around the folded protein, involving at least two defined regions of the PfRH5 primary sequence; iii) a brief exposure windows of PfRH5 is likely to necessitate rapid binding of antibody to neutralize parasites; and iv) 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 antibodies and further encourage anti-PfRH5 based malaria prevention efforts. INTRODUCTION Despite recent progress in malaria control, current estimates of deaths per year from contamination remain unacceptably high (1). The Gemcitabine prospects of artemisinin-resistant parasites and pyrethroid-resistant mosquitoes mandate on-going research into novel cost-effective interventions to control apical membrane antigen 1 (PfAMA1) and merozoite surface protein 2 (PfMSP2)-based vaccines (4, 5). Strain-specific efficacy has also been apparent with PfAMA1 and PfMSP1 vaccines in non-human primate models (6, 7). We recently reported that vaccines based upon the antigen reticulocyte-binding protein homologue 5 (PfRH5) were capable of inducing antibodies that neutralized multiple parasite laboratory lines, as well as recently culture-adapted field isolates, in the widely used assay of growth inhibitory activity (GIA) (8-10). Quite unlike previous blood-stage vaccine candidate antigens, PfRH5 does not appear to be a major target of naturally-acquired immunity to (8, 11). Moreover, PfRH5 is usually highly conserved across parasite lines, with only five non-synonymous SNPs identified at frequencies 5% in at least one geographical region and among 227 sequenced field parasite isolates (9, 10). Merozoite invasion of erythrocytes is Gemcitabine Rabbit Polyclonal to PTX3 usually a complex process involving a series Gemcitabine of actions, proceeding through initial binding, reorientation, and committed attachment, followed by moving junction (MJ) motility and vacuole formation (12). Among other functions, proteins around the merozoite surface or secreted from the apical organelles mediate binding to host receptors and/or trigger subsequent actions of invasion through poorly defined signal transduction mechanisms (13-15). Such proteins are accessible to antibody which may interfere with these functions and hence inhibit invasion. The conversation of PfRH5 with the erythrocyte surface protein basigin Gemcitabine (BSG/CD147) is essential for merozoite invasion into erythrocytes, and blockade of this conversation by monoclonal antibodies (mAbs) to the BSG host receptor can inhibit invasion (16). Although it is usually tempting to speculate that this mechanism of action of vaccine-induced anti-PfRH5 antibody may be comparable, previous studies of antibodies against other blood-stage antigens have identified rather more complex and mixed mechanisms of action. For example, there appear to be at least three distinct mechanisms of action of antibody against PfMSP1 (17); whilst a recent study found that two anti-PfAMA1 mAbs act via blockade of the conversation of PfAMA1 with rhoptry neck protein 2 (PfRON2) (18), but additional actions of polyclonal anti-PfAMA1 antibodies are likely (19). Although we have shown that antibodies against PfRH5 can effectively neutralize parasites, no more detailed description has yet been provided of the mechanism of action of these antibodies. Here, we characterize the effect of polyclonal antibodies against PfRH5, and report the development of a panel of mouse mAbs which we describe in detail, including epitope and kinetic analyses. Although we demonstrate that blockade of the PfRH5-BSG conversation by anti-PfRH5 mAbs consistently results in merozoite neutralization, we report further results suggesting additional actions may contribute to parasite neutralization by anti-PfRH5 antibody. METHODS Vaccines, animals and polyclonal antibody generation BALB/c mice and New Zealand white rabbits were immunized intramuscularly (i.m.) with human adenovirus serotype 5 (AdHu5) and altered vaccinia computer virus Ankara (MVA) viral vectors at an 8 to 17 week interval, expressing full-length PfRH5 (3D7 allele); a PfRH5 fragment (amino acids 191 C 359, NSIY IRYH); a bi-allelic PfAMA1 insert; or no malaria antigen as previously described (8). The latter control immunizations of mice used viruses expressing ovalbumin (20), Gemcitabine whereas rabbits were immunized with viruses lacking an antigen.