Malaria Vaccine Development Research Articles

Steady progress toward a malaria vaccine.

Great progress has been made in reducing malaria morbidity and mortality, yet the parasite continues to cause a startling 200 million infections and 500 000 deaths annually. Malaria vaccine development is pushing new boundaries by steady advancement toward a licensed product. Despite 50 years of research, the complexity of Plasmoidum falciparum confounds all attempts to eradicate the organism. This very complexity has pushed the boundaries of vaccine development to new heights, yet it remains to be seen if an affordable vaccine can provide durable and high-level protection. Novel vaccines such as RTS,S/AS01E are on the edge of licensure, but old techniques have resurged with the ability to deliver vialed, whole organism vaccines. Novel adjuvants, multistage/multiantigen approaches and transmission blocking vaccines all contribute to a multipronged battle plan to conquer malaria. Vaccines are the most cost-effective tools to control infectious diseases, yet the complexity of malaria has frustrated all attempts to develop an effective product. This review concentrates on recent advances in malaria vaccine development that lend hope that a vaccine can be produced and malaria eradicated.

The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum

BackgroundAt a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study.ResultsA pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-119, MSP-142, MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule.ConclusionAnalysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross-comparisons of results of vaccine trials performed in different centres/with different products; (2) to facilitate standardization and harmonization of immunological assays used in epidemiology research; and (3) to allow optimization and validation of immunological assays used in malaria vaccine development.

Structural analysis of owl monkey MHC-DR shows that fully-protective malaria vaccine components can be readily used in humans

More than 50 years ago the owl monkey (genus Aotus) was found to be highly susceptible to developing human malaria, making it an excellent experimental model for this disease. Microbes and parasites' (especially malaria) tremendous genetic variability became resolved during our malaria vaccine development, involving conserved peptides having high host cell binding activity (cHABPs); however, cHABPs are immunologically silent and must be specially modified (mHABPs) to induce a perfect fit into major histocompatibility complex (MHC) molecules (HLA in humans). Since malarial immunity is mainly antibody-mediated and controlled by the HLA-DRB genetic region, ∼1000 Aotus have been molecularly characterised for MHC-DRB, revealing striking similarity between human and Aotus MHC-DRB repertories. Such convergence suggested that a large group of immune protection-inducing protein structures (IMPIPS), highly immunogenic and protection inducers against malarial intravenous challenge in Aotus, could easily be used in humans for inducing full protection against malaria. We highlight the value of a logical and rational methodology for developing a vaccine in an appropriate animal model: Aotus monkeys.

Dynamics in multiplicity of Plasmodium falciparum infection among children with asymptomatic malaria in central Ghana

BackgroundThe determinants of malaria parasite virulence is not entirely known, but the outcome of malaria infection (asymptomatic or symptomatic) has been associated with carriage of distinct parasite genotypes. Alleles considered important for erythrocyte invasion and selected as candidate targets for malaria vaccine development are increasingly being shown to have distinct characteristics in infection outcomes. Any unique/distinct patterns or alleles linked to infection outcome should be reproducible for a given malaria-cohort regardless of location, time or intervention. This study compared merozoite surface protein 2 (MSP2) genotypes from children with asymptomatic malaria at same geographical location, from two time periods.ResultsAs the prevalence and incidence of malaria (measured for other studies) significantly reduced between 2004 (time point one) and 2009 (time point two), MSP2 multiplicity of infections (MOI) also reduced significantly from 2.3 at time point (TP) one to 1.9 at TP two. IC/3D7 genotypes out-numbered FC27 genotypes at both time points. At TP2 however, FC27 allele diversity was more than the IC/3D7 allele diversity. A decrease in the IC/3D7:FC27 genotype proportions from 2:1 at TP1 to 1:1 at TP2, seemed to be driven mainly by a decrease in carriage of IC/3D7 alleles. MOI was higher in the dry season than in the subsequent wet season, but the decrease was not significant at TP2.ConclusionMSP2 MOI was higher in the dry season than in the subsequent wet season, while the carriage of IC/3D7 alleles decreased over this time period. It may be that decreases in transmission are related specifically to the IC/3D7 allelic family. The influence of transmission on MSP2 allele diversity needs to be clearly deciphered in studies which should include the use of sensitive methods for the detection of polymorphic parasite markers for both symptomatic and asymptomatic malaria. Such studies will enable better understanding of associations between allelic variants, MOI, transmission, malaria infection and disease.

Enhanced acquired antibodies to a chimeric Plasmodium falciparum antigen; UB05-09 is associated with protective immunity against malaria.

SummaryIt has been shown that covalently linking two antigens could enhance the immunogenicity of the chimeric construct. To prioritize such a chimera for malaria vaccine development, it is necessary to demonstrate that naturally acquired antibodies against the chimera are associated with protection from malaria. Here, we probe the ability of a chimeric construct of UB05 and UB09 antigens (UB05‐09) to better differentiate between acquired immune protection and susceptibility to malaria. In a cross‐sectional study, recombinant UB05‐09 chimera and the constituent antigens were used to probe for specific antibodies in the plasma from children and adults resident in a malaria‐endemic zone, using the enzyme‐linked immunosorbent assay (ELISA). Anti‐UB05‐09 antibody levels doubled that of its constituent antigens, UB09 and UB05, and this correlated with protection against malaria. The presence of enhanced UB05‐09‐specific antibody correlated with the absence of fever and parasitaemia, which are the main symptoms of malaria infection. The chimera is more effective in detecting and distinguishing acquired protective immunity against malaria than any of its constituents taken alone. Online B‐cell epitope prediction tools confirmed the presence of B‐cell epitopes in the study antigens. UB05‐09 chimera is a marker of protective immunity against malaria that needs to be studied further.

Chemical Attenuation in the Development of a Whole-Organism Malaria Vaccine.

Malaria vaccine development has been dominated by the subunit approach; however, many subunit vaccine candidates have had limited efficacy in settings of malaria endemicity. As our search for an efficacious malaria vaccine continues, the development of a whole-organism vaccine is now receiving much scrutiny. One strategy currently being explored in the development of a whole-organism vaccine involves chemical attenuation of the malaria parasite. In vivo and in vitro chemical attenuation of both liver-stage and blood-stage Plasmodium parasites has been investigated. Here, we discuss both approaches of chemical attenuation in the development of a whole-organism vaccine against malaria.

Limited genetic diversity in the global Plasmodium vivax Cell traversal protein of Ookinetes and Sporozoites (CelTOS) sequences; implications for PvCelTOS-based vaccine development

Cell traversal protein of Ookinetes and Sporozoites (CelTOS) is a new malaria vaccine candidate antigen. Since one of the main challenges in malaria vaccine development is the extensive antigenic diversity of this parasite, local and global gene diversity analysis is of particular importance. Therefore, in this study, the genetic diversity of pvceltos gene was investigated among Iranian P. vivax isolates (n=46) and compared with available worldwide pvceltos sequences. One synonymous (C109A) and three amino acid replacements (V118L, K178T, and G179R) were observed in Iranian pvceltos sequences in compare with Sal-1 sequence leading to five haplotypes including PvCelt-A (GSVKGL, 13%), PvCelt-B (GSLKGL, 50%), PvCelt-C (GSLTGL, 17.4%), PvCelt-D (GSVTGL, 13%) and PvCelt-E (GSLTRL, 6.5%). However, amino acid replacements were observed in six positions (G10S, S40N, V118L/M, K178T, G179R/D and L181R) in PvCelTOS antigen of global isolates leading to 11 distinct haplotypes. PvCelt-A and PvCelt-B haplotypes were the most common haplotypes in the world. The overall nucleotide diversity for Iranian isolates was 0.00169, while, the level of nucleotide diversity was ranged from 0.00252 for Thailand to 0.00022 for Peru populations in the world. The analysis of SNPs in relation with the predicted immunodominant regions revealed that only K178T and G179R SNPs are located in putative B-cell epitopes. All replacements were located in CD4+ and/or CD8+ T-cell epitopes. However, the majority of epitopes are located in conserved regions. Knowing whether these changes may alter the affinity of the epitopes for antibodies and/or MHC molecules remains to be investigated in experimental studies. In conclusion, the present study showed a very limited genetic diversity in pvceltos gene among the global clinical isolates that can be regarded as a potential candidate antigen to apply for vivax-based malaria vaccine development.

Vaccines against malaria—still a long way to go

Several species of Plasmodium cause a broad spectrum of human disease that range from nausea and fever to severe anemia, cerebral malaria, and multiorgan failure. In malaria-endemic countries, continuous exposure to Plasmodium sporozoite inoculations and subsequent blood infections elicit only partial and short-lived immunity, which gradually develops over many years of parasite exposure and multiple clinical episodes. The ambitious goal of malaria vaccinology over the past 70 years has been to develop an immunization strategy that mounts protection superior to naturally acquired immunity. Herein, three principal concepts in evidence-based malaria vaccine development are compared. Feasible leads are typically stand-alone subunit vaccine approaches that block Plasmodium parasite life cycle progression or parasite/host interactions, and they constitute the majority of candidates in preclinical research and early clinical testing. Integrated approaches incorporate malaria antigen(s) into licensed or emerging pediatric vaccine formulations. This strategy can complement the malaria control portfolio even if the antimalarial component is only partially effective and has led to the development of the only candidate vaccine to date, namely RTS,S-AS01. Experimental whole parasite vaccine approaches have been repeatedly shown to elicit sterile and lasting protection against identical parasite strains, but mass production, proof of broad protection against different parasite strains, and routes of vaccine delivery remain significant translational road blocks. Global access to an effective and affordable malaria vaccine will critically depend on innovative translational research that builds on a better molecular understanding of Plasmodium biology and host immunity.

Critical role of HLA-DRβ* binding peptides' peripheral flanking residues in fully-protective malaria vaccine development

A vaccine candidate component must fit perfectly into the antigen presenting HLA-DRβ* molecule's groove (or canonical nonapeptide) peptide binding region (PBR) during antigen presentation to the T-cell receptor (TCR), conforming a specific and stable macromolecular complex and induce an appropriate immune response.Antigen's peripheral flanking residues (PFR, positions (p) -p2 and p10) must thus establish strong interactions with the HLA-DRβ* - TCR complex. These amino acids (aa) have specific physico-chemical characteristics enabling differentiation between non-protective but antibody-inducer (NPAI), short-lived protection inducer (SLPI) and long-lasting protection inducer (LLPI) peptides when used as an antimalarial vaccine component. Their identification (through 1H-NMR and Aotus monkey immunization) and proper modification contributes to a logical and rational methodology for long-lasting and protective immunological memory.

Role of controlled human malaria infection (CHMI) in malaria vaccine development: A U.S. food & drug administration (FDA) perspective

Role of controlled human malaria infection (CHMI) in malaria vaccine development: A U.S. food & drug administration (FDA) perspective

Naturally Acquired Antibodies Target the Glutamate-Rich Protein on Intact Merozoites and Predict Protection Against Febrile Malaria.

Plasmodium species antigens accessible at the time of merozoite release are likely targets of biologically functional antibodies. Immunoglobulin G (IgG) antibodies against intact merozoites were quantified in the plasma of Ghanaian children from a longitudinal cohort using a novel flow cytometry-based immunofluorescence assay. Functionality of these antibodies, as well as glutamate-rich protein (GLURP)-specific affinity-purified IgG from malaria hyperimmune Liberian adults, was assessed by the opsonic phagocytosis (OP) assay. Opsonic phagocytosis activity was strongly associated (hazard ratio [HR] = 0.46; 95% confidence interval [CI] = .30-.73; P = .0008) with protection against febrile malaria. Of the antimerozoite-specific antibodies, only IgG3 was significantly associated with both OP and protection (HR = 0.53; 95% CI = .34-.84; Pcorrected = .03) against febrile malaria. Similarly, GLURP-specific antibodies previously shown to be protective against febrile malaria in this same cohort were significantly associated with OP activity in this study. GLURP-specific antibodies recognized merozoites and also mediated OP activity. These findings support previous studies that found OP of merozoites to be associated with protection against malaria and further shows IgG3 and GLURP antibodies are key in the OP mechanism, thus giving further impetus for the development of malaria vaccines targeting GLURP.

Magnetic Nanovectors for the Development of DNA Blood-Stage Malaria Vaccines.

DNA vaccines offer cost, flexibility, and stability advantages, but administered alone have limited immunogenicity. Previously, we identified optimal configurations of magnetic vectors comprising superparamagnetic iron oxide nanoparticles (SPIONs), polyethylenimine (PEI), and hyaluronic acid (HA) to deliver malaria DNA encoding Plasmodium yoelii (Py) merozoite surface protein MSP119 (SPIONs/PEI/DNA + HA gene complex) to dendritic cells and transfect them with high efficiency in vitro. Herein, we evaluate their immunogenicity in vivo by administering these potential vaccine complexes into BALB/c mice. The complexes induced antibodies against PyMSP119, with higher responses induced intraperitoneally than intramuscularly, and antibody levels further enhanced by applying an external magnetic field. The predominant IgG subclasses induced were IgG2a followed by IgG1 and IgG2b. The complexes further elicited high levels of interferon gamma (IFN-γ), and moderate levels of interleukin (IL)-4 and IL-17 antigen-specific splenocytes, indicating induction of T helper 1 (Th1), Th2, and Th17 cell mediated immunity. The ability of such DNA/nanoparticle complexes to induce cytophilic antibodies together with broad spectrum cellular immunity may benefit malaria vaccines.

THE QUEST FOR BUILDING LABORATORY CAPACITY TO SUPPORT CONTROLLED HUMAN MALARIA INFECTION (CHMI) STUDIES IN SUB-SAHARAN AFRICA: EXPERIENCE WITH FIVE SITES

BackgroundConducting Controlled Human Malaria Infection (CHMI) studies in sub-Saharan Africa presents unique challenges yet it provides enormous opportunities for fast-tracking malaria vaccine and drug development. Sanaria Inc. USA, has devised...

Biophysical and Structural Characterization of Antibody Responses to Malaria Antigens

Malaria is a global health priority: 214 million malaria cases were reported in 2015 alone, predominantly in Africa and resulting in 438,000 deaths - 70% of which were in children below five years of age. Reverse vaccinology holds promise to design effective immunogens for the development of malaria vaccines. This concept is based on interrogating the B cell repertoire of vaccinated or infected subjects to identify protective antibodies that will guide immunogen design. Our efforts have focused on plasmodium falciparum targets associated with the development of pre-erythrocytic and transmission-blocking vaccines. We isolated B cells from individuals naturally exposed to plasmodium falciparum, and from vaccinated animals and humans having undergone controlled infection. We performed extensive binding experiments to characterize affinities and competition of dozens of antibodies by isothermal titration calorimetry and biolayer interferometry. These studies uncovered several epitope bins, which give biophysical insights into immunodominant and protective B cell responses. X-ray crystallography, small-angle X-ray scattering and single-particle electron microscopy were used and integrated to define antibody recognition at the molecular level. Our structural delineation of protective epitopes provides the blueprints to engineer optimized antigens that can be formulated and tested as pre-erythrocytic and transmission-blocking malaria vaccines.

Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal ‘C-tag’

Development of bespoke biomanufacturing processes remains a critical bottleneck for translational studies, in particular when modest quantities of a novel product are required for proof-of-concept Phase I/II clinical trials. In these instances the ability to develop a biomanufacturing process quickly and relatively cheaply, without risk to product quality or safety, provides a great advantage by allowing new antigens or concepts in immunogen design to more rapidly enter human testing. These challenges with production and purification are particularly apparent when developing recombinant protein-based vaccines for difficult parasitic diseases, with Plasmodium falciparum malaria being a prime example. To that end, we have previously reported the expression of a novel protein vaccine for malaria using the ExpreS2Drosophila melanogaster Schneider 2 stable cell line system, however, a very low overall process yield (typically <5% recovery of hexa-histidine-tagged protein) meant the initial purification strategy was not suitable for scale-up and clinical biomanufacture of such a vaccine. Here we describe a newly available affinity purification method that was ideally suited to purification of the same protein which encodes the P. falciparum reticulocyte-binding protein homolog 5 – currently the leading antigen for assessment in next generation vaccines aiming to prevent red blood cell invasion by the blood-stage parasite. This purification system makes use of a C-terminal tag known as ‘C-tag’, composed of the four amino acids, glutamic acid – proline – glutamic acid – alanine (E-P-E-A), which is selectively purified on a CaptureSelect™ affinity resin coupled to a camelid single chain antibody, called NbSyn2. The C-terminal fusion of this short C-tag to P. falciparum reticulocyte-binding protein homolog 5 achieved >85% recovery and >70% purity in a single step purification directly from clarified, concentrated Schneider 2 cell supernatant under mild conditions. Biochemical and immunological analysis showed that the C-tagged and hexa-histidine-tagged P. falciparum reticulocyte-binding protein homolog 5 proteins are comparable. The C-tag technology has the potential to form the basis of a current good manufacturing practice-compliant platform, which could greatly improve the speed and ease with which novel protein-based products progress to clinical testing.

The role of pi-interactions and hydrogen bonds in fully protective synthetic malaria vaccine development

Analysis of our Plasmodium falciparum malaria parasite peptides' 1H-NMR database in the search for H-bonds and π-interactions led us to correlate their presence or absence with a peptide's particular immunological behavior. It was concluded that a 26.5 ± 1.5 Å between positions 1 to 9 of the HLA-DRβ1* interacting region was necessary for proper docking of 20mer-long peptides and these MHC Class II molecules for full-protective immunity. Presence of intramolecular H-bonds or π-interactions leading to righ-handed α-helix or β-turn conformation in this peptide's region induces different immune responses or none. PPIIL conformation and the absence of any intramolecular interaction thus became the first feature characterising our immune protection-inducing structures as malaria vaccine candidates.

The Power of Malaria Vaccine Trials Using Controlled Human Malaria Infection.

Controlled human malaria infection (CHMI) in healthy human volunteers is an important and powerful tool in clinical malaria vaccine development. However, power calculations are essential to obtain meaningful estimates of protective efficacy, while minimizing the risk of adverse events. To optimize power calculations for CHMI-based malaria vaccine trials, we developed a novel non-linear statistical model for parasite kinetics as measured by qPCR, using data from mosquito-based CHMI experiments in 57 individuals. We robustly account for important sources of variation between and within individuals using a Bayesian framework. Study power is most dependent on the number of individuals in each treatment arm; inter-individual variation in vaccine efficacy and the number of blood samples taken per day matter relatively little. Due to high inter-individual variation in the number of first-generation parasites, hepatic vaccine trials required significantly more study subjects than erythrocytic vaccine trials. We provide power calculations for hypothetical malaria vaccine trials of various designs and conclude that so far, power calculations have been overly optimistic. We further illustrate how upcoming techniques like needle-injected CHMI may reduce required sample sizes.

Pro-inflammatory Cytokine Response and Genetic Diversity in Merozoite Surface Protein 2 of Plasmodium falciparum Isolates from Nigeria.

Background:Polymorphisms in Plasmodium falciparum merozoite surface protein-2 (msp-2) and associated parasite genetic diversity which varies between malaria-endemic regions remain a limitation in malaria vaccine development. Pro-inflammatory cytokines are important in immunity against malaria, understanding the influence of genetic diversity on cytokine response is important for effective vaccine design.Methods:P. falciparum isolates obtained from 300 Nigerians with uncomplicated falciparum malaria at Ijede General Hospital, Ijede (IJE), General Hospital Ajeromi, Ajeromi (AJE) and Saint Kizito Mission Hospital, Lekki, were genotyped by nested polymerase chain reaction of msp-2 block 3 while ELISA was used to determine the pro-inflammatory cytokine response to describe the genetic diversity of P. falciparum.Results:Eighteen alleles were observed for msp-2 loci. Of the 195 isolates, 61 (31.0%) had only FC27-type alleles, 38 (19.7%) had only 3D7-type alleles, and 49.3% had multiple parasite lines with both alleles. Band sizes were 275–625 bp for FC27 and 150–425 bp for 3D7. Four alleles were observed from LEK, 2 (375–425 bp) and 2 (275–325 bp) of FC27-and 3D7-types, respectively; 12 alleles from AJE, 9 (275–625 bp) and 3 (325–425 bp) of FC27-types and 3D7-types, respectively; while IJE had a total of 12 alleles, 9 (275–625 bp) and 3 (325–425 bp) of FC27-types and 3D7-types, respectively. Mean multiplicity of infection (MOI) was 1.54. Heterozygosity (HE) ranged from 0.77 to 0.87 and was highest for IJE (0.87). Cytokine response was higher among <5 years and was significantly associated with MOI (P > 0.05) but with neither parasite density nor infection type.Conclusion:P. falciparum genetic diversity is extensive in Nigeria, protection via pro-inflammatory cytokines have little or no interplay with infection multiplicity.

Naturally Acquired Antibody Responses to a Synthetic Malaria Antigen AS202.11.

Background A major challenge to malaria vaccine development is identification of protective epitopes and respective protective immune responses. Objective To characterize naturally acquired Immunoglobulin G (IgG) responses to the synthetic peptide AS202.11, a malaria vaccine candidate. Methodology This community based cross-sectional study enrolled 320 participants aged 1 year and above. Demographic information was recorded through interviews. Detection of P. falciparum infection was done by microscopy, malaria rapid diagnostic test, and polymerase chain reaction. ELISA was used to detect IgG antibody. Data was analyzed using STATA. Results The overall AS202.11 IgG seropositivity was 78.8% (73.9–82.9). Seropositivity by age categories was ≤12 years [74.3% (67.4–80.2)], 13–40 years [85.3% (76.5–91.1)], and >40 years [82.6% (68.7–91.1)]. Compared to the ≤ 12-year-old group, aORs for the other groups were 2.22 (1.14–4.32), p = 0.019, and 1.87 (0.81–4.35), p = 0.143, for the 13–40-year-old and >40-year-old groups, respectively. The 13–40-year-old group had more seropositive individuals compared to the ≤ 12-year-old group. Conclusion We report a high degree of recognition of AS202.11 by IgG elicited by field P. falciparum strains, suggesting its close similarity to native P. falciparum antigens and possible suitability of the peptide as a future malaria vaccine candidate.

Comparison of functional assays used in the clinical development of a placental malaria vaccine

BackgroundMalaria in pregnancy is associated with significant morbidity in pregnant women and their offspring. Plasmodium falciparum infected erythrocytes (IE) express VAR2CSA that mediates binding to chondroitin sulphate A (CSA) in the placenta. Two VAR2CSA-based vaccines for placental malaria are in clinical development. The purpose of this study was to evaluate the robustness and comparability of binding inhibition assays used in the clinical development of placental malaria vaccines. MethodsThe ability of sera from animals immunised with different VAR2CSA constructs to inhibit IE binding to CSA was investigated in three in vitro assays using 96-well plates, petri dishes, capillary flow and an ex vivo placental perfusion assay. ResultsThe inter-assay variation was not uniform between assays and ranged from above ten-fold in the flow assay to two-fold in the perfusion assay. The intra-assay variation was highest in the petri dish assay. A positive correlation between IE binding avidity and the level of binding after antibody inhibition in the petri dish assay indicate that high avidity IE binding is more difficult to inhibit. The highest binding inhibition sensitivity was found in the 96-well and petri dish assays compared to the flow and perfusion assays where binding inhibition required higher antibody titers. ConclusionsThe inhibitory capacity of antibodies is not easily translated between assays and the high sensitivity of the 96-well and petri dish assays stresses the need for comparing serial dilutions of serum. Furthermore, IE binding avidity must be in the same range when comparing data from different days. There was an overall concordance in the capacity of antibody-mediated inhibition, when comparing the in vitro assays with the perfusion assay, which more closely represents in vivo conditions. Importantly the ID1-ID2a protein in a liposomal formulation, currently in a phase I trial, effectively induced antibodies that inhibited IE adhesion in placental tissue.