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Background: Overcoming antigenic diversity is a key challenge in the development of effective Plasmodium falciparum malaria vaccines. Strategies that promote the generation of antibodies targeting conserved epitopes of vaccine antigens may provide protection against diverse parasites strains. Understanding differences between vaccine-induced and naturally acquired immunity is important to achieving this goal. Methods: We analyzed antibodies generated in a phase 1 human vaccine trial, MSP2-C1, which included 2 allelic forms of MSP2, an abundant vaccine antigen on the merozoite surface. Vaccine-induced responses were assessed for functional activity against multiple parasite strains, and cross-reactivity of antibodies was determined using competition ELISA and epitope mapping approaches. Results: Vaccination induced cytophilic antibody responses with strain-transcending opsonic phagocytosis and complement-fixing function. In contrast to antibodies acquired via natural infection, vaccine-induced antibodies were directed towards conserved epitopes at the C-terminus of MSP2, whereas naturally acquired antibodies mainly targeted polymorphic epitopes. Functional activity of C-terminal–targeted antibodies was confirmed using monoclonal antibodies that promoted opsonic phagocytosis against multiple parasite strains. Conclusion: Vaccination generated markedly different responses to polymorphic antigens than naturally acquired immunity and targeted conserved functional epitopes. Induction of antibodies targeting conserved regions of malaria antigens provides a promising vaccine strategy to overcome antigenic diversity for developing effective malaria vaccines.

Invasion of human erythrocytes is essential for Plasmodium falciparum parasite survival and pathogenesis, and is also a complex phenotype. While some later steps in invasion appear to be invariant and essential, the earlier steps of recognition are controlled by a series of redundant, and only partially understood, receptor-ligand interactions. Reverse genetic analysis of laboratory adapted strains has identified multiple genes that when deleted can alter invasion, but how the relative contributions of each gene translate to the phenotypes of clinical isolates is far from clear. We used a forward genetic approach to identify genes responsible for variable erythrocyte invasion by phenotyping the parents and progeny of previously generated experimental genetic crosses. Linkage analysis using whole genome sequencing data revealed a single major locus was responsible for the majority of phenotypic variation in two invasion pathways. This locus contained the PfRh2a and PfRh2b genes, members of one of the major invasion ligand gene families, but not widely thought to play such a prominent role in specifying invasion phenotypes. Variation in invasion pathways was linked to significant differences in PfRh2a and PfRh2b expression between parasite lines, and their role in specifying alternative invasion was confirmed by CRISPR-Cas9-mediated genome editing. Expansion of the analysis to a large set of clinical P. falciparum isolates revealed common deletions, suggesting that variation at this locus is a major cause of invasion phenotypic variation in the endemic setting. This work has implications for blood-stage vaccine development and will help inform the design and location of future large-scale studies of invasion in clinical isolates., Author summary Plasmodium parasites cause more than 200 million cases of malaria each year. All the symptoms of malaria are caused after Plasmodium parasites invade human red blood cells. Once inside, they grow, multiply and break open the red blood cells to release new parasites. This cycle is repeated every 48 hours, rapidly amplifying the number of parasites and causing severe anemia and other complications. Plasmodium falciparum, the parasite species responsible for almost all malaria deaths, can use multiple different pathways to invade human red blood cells, but the relative importance of each is not well understood. We tested the invasion pathways used by a collection of closely related parasites and compared their genome sequences to identify the genes responsible. This analysis revealed that expression differences in two neighboring genes of the Reticulocyte Binding Homologue family are responsible for most of the variation in two invasion pathways. P. falciparum may use variation in these genes to avoid the immune system or adapt to specific blood groups, which has important implications for vaccine development against malaria.

Malaria has been one of the strongest selective forces on the human genome. The increased frequency of haemoglobinopathies, as well as numerous other blood groups, in malaria endemic regions is commonly attributed to a protective effect of these alleles against malaria. In the majority of these cases however there have been no systematic functional studies to test protective mechanisms, in large part because most host-parasite interaction assays are not quantitative or scalable. We describe the development of an erythrocyte preference assay which uses differential labelling with fluorescent dyes to distinguish invasion into four different erythrocyte populations which are all co-incubated with a single Plasmodium falciparum parasite culture. Testing this assay on erythrocytes across the ABO blood system from forty independent donors reveals for the first time that P. falciparum parasites preferentially invade group O over Group A erythrocytes. This runs counter to the known protective effect of group O against severe malaria, but emphasises the complexities of host-pathogen interactions, and the need for highly quantitative and scalable assays to systematically explore them.

Significance Malaria still kills hundreds of thousands of children each year. Malaria vaccine development is complicated by high levels of parasite genetic diversity, which makes single target vaccines vulnerable to the development of variant-specific immunity. To overcome this hurdle, we systematically screened a panel of 29 blood-stage antigens from the most deadly human malaria parasite, Plasmodium falciparum. We identified several targets that were able to inhibit erythrocyte invasion in two genetically diverse strains. Testing these targets in combination identified several pairs that blocked invasion more effectively in combination than in isolation. Video microscopy and studies of natural immune responses to malaria in patients suggest that targeting multiple steps in invasion is more likely to produce a synergistic vaccine response., A highly effective vaccine would be a valuable weapon in the drive toward malaria elimination. No such vaccine currently exists, and only a handful of the hundreds of potential candidates in the parasite genome have been evaluated. In this study, we systematically evaluated 29 antigens likely to be involved in erythrocyte invasion, an essential developmental stage during which the malaria parasite is vulnerable to antibody-mediated inhibition. Testing antigens alone and in combination identified several strain-transcending targets that had synergistic combinatorial effects in vitro, while studies in an endemic population revealed that combinations of the same antigens were associated with protection from febrile malaria. Video microscopy established that the most effective combinations targeted multiple discrete stages of invasion, suggesting a mechanistic explanation for synergy. Overall, this study both identifies specific antigen combinations for high-priority clinical testing and establishes a generalizable approach that is more likely to produce effective vaccines.

The development of effective malaria vaccines and immune biomarkers of malaria is a high priority for malaria control and elimination. Ags expressed by merozoites of Plasmodium falciparum are likely to be important targets of human immunity and are promising vaccine candidates, but very few Ags have been studied. We developed an approach to assess Ab responses to a comprehensive repertoire of merozoite proteins and investigate whether they are targets of protective Abs. We expressed 91 recombinant proteins, located on the merozoite surface or within invasion organelles, and screened them for quality and reactivity to human Abs. Subsequently, Abs to 46 proteins were studied in a longitudinal cohort of 206 Papua New Guinean children to define Ab acquisition and associations with protective immunity. Ab responses were higher among older children and those with active parasitemia. High-level Ab responses to rhoptry and microneme proteins that function in erythrocyte invasion were identified as being most strongly associated with protective immunity compared with other Ags. Additionally, Abs to new or understudied Ags were more strongly associated with protection than were Abs to current vaccine candidates that have progressed to phase 1 or 2 vaccine trials. Combinations of Ab responses were identified that were more strongly associated with protective immunity than responses to their single-Ag components. This study identifies Ags that are likely to be key targets of protective human immunity and facilitates the prioritization of Ags for further evaluation as vaccine candidates and/or for use as biomarkers of immunity in malaria surveillance and control.

How the immune system responds to exposure to infections during pregnancy is poorly understood. Antibody-mediated immunity is essential for protection against many important pathogens, but how antibodies are acquired, maintained, and boosted after re-exposure during pregnancy remains unknown. This knowledge is critical to understanding why pregnant women are more susceptible to and more severely affected by numerous infectious diseases [1] and has translational implications for the development and use of vaccines to protect pregnant women and their babies. In this study, we aimed to address these key questions by studying responses to one of the most important human pathogens during pregnancy: malaria. It is estimated that every year 125 million women living in areas where malaria is endemic become pregnant [2]. Infection with Plasmodium falciparum and Plasmodium vivax during pregnancy contributes to maternal anemia, can cause severe illness and death, and leads to low infant birth weight, which is a major risk factor for infant mortality and morbidity [3, 4]. At the time of their first pregnancy, women living in areas where malaria is endemic may have developed substantial acquired immunity to malaria, which does not prevent infection per se, but controls high-density parasitemia and associated clinical symptoms [5]. Antibodies against the disease-causing blood stage of malaria have a significant role in protection and target antigens on the surface of merozoites and infected erythrocytes (IE) [6, 7]. Despite pre-existing immunity, pregnant women develop placental and peripheral infections at higher parasite densities, compared with nonpregnant adults [8]. This susceptibility has been attributed to immune modulation resulting in an impaired ability to limit parasite replication during pregnancy and the emergence of specific antigenic variants of P. falciparum that evade existing immunity and accumulate in the placenta [9, 10]. The expression by P. falciparum IEs of the VAR2CSA protein, a specific variant of P. falciparum erythrocyte membrane protein (PfEMP1) that is exposed on the surface of IEs, facilitates the sequestration of IEs in the placenta by mediating adhesion to chondroitin sulfate A and, possibly, other receptors in the intervillous space [9–11]. Levels of antibodies to surface antigens of placental-binding IEs, and VAR2CSA specifically, are generally low before pregnancy and are higher in multigravida women exposed to P. falciparum [9–12]. Little is known about the maintenance and boosting of antimalarial responses over time, particularly during pregnancy, and there is a paucity of studies with repeated sampling over time or studies examining responses to multiple infections. Furthermore, very little is known about antibody responses to non–P. falciparum malaria during pregnancy, particularly P. vivax, which is widespread in Asia and South America. To advance our knowledge on the acquisition and boosting of antibody responses to infections in pregnant women and the maintenance of antibodies in the absence of reinfection, we studied antimalarial responses throughout pregnancy among women exposed to P. falciparum and P. vivax infection in a region of Southeast Asia where malaria is endemic.

BACKGROUND: Metastatic germ cell tumors remain potentially curable when treated with salvage chemotherapy at first relapse. In the present phase I/II study, we sought to improve on the response rate and duration of the TIP (paclitaxel, ifosfamide, cisplatin) regimen by adding gemcitabine (Gem-TIP).MATERIALS AND METHODS: Twenty patients were recruited after failure of first-line cisplatin-containing chemotherapy. The primary objectives were to determine the maximum tolerated dose of gemcitabine when combined with TIP and to establish the dose intensity of the TIP drugs in this combination. The secondary objectives were the response rates, failure-free survival, and overall survival.RESULTS: The maximum tolerated dose of gemcitabine was 1200 mg/m2. The mean relative dose intensity was 95% (95% confidence interval [CI], 90.2%-99.2%) for gemcitabine, 96% (95% CI, 92.9%-98.7%) for paclitaxel, 92% (95% CI, 84.5%-98.8%) for ifosfamide, and 94% (95% CI, 89.3%-99.0%) for cisplatin. The overall complete response rate was 50%; another 30% of the patients achieved a partial response. The 1-year failure-free survival and overall survival rates were 68% (95% CI, 43%-84%) and 89.5% (95% CI, 64%-97%), respectively.CONCLUSION: Gemcitabine can be added to TIP chemotherapy at the full dose, with manageable toxicity and no detrimental effect on the dose intensity of the TIP drugs. The response rate and duration were improved compared with those reported from the Medical Research Council TIP trial; further evaluation is warranted.

Background Malaria is a major global cause of deaths and a vaccine is urgently needed. Results We have employed the P. falciparum merozoite antigens MSP2-3D7/FC27 and AMA1, used them in ELISA, and coupled them in different ways using surface plasmon resonance (SPR) and estimated affinity (measured as kd) of monoclonal as well as naturally-acquired polyclonal antibodies in human plasma. There were major differences in kd depending on how the antigens were immobilized and where the His-tag was placed. For AMA1 we could see correlations with invasion inhibition. Using different immobilizations of proteins in SPR, we could see only moderate correlations with levels of antibodies in ELISA, indicating that in ELISA the proteins were not uniformly bound and that antibodies with many specificities exist in natural immunisation. The correlations between ELISA and SPR were enhanced when only parasite positive samples were included, which may indicate that high affinity antibodies are difficult to maintain over long periods of time. We found higher kd values for MSP2 (indicating lower affinity) compared to AMA1, which might be partly explained by MSP2 being an intrinsically disordered protein, while AMA1 is globular. Conclusions For future vaccine studies and for understanding immunity, it is important to consider how to present proteins to the immune system to achieve highest antibody affinities. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0461-1) contains supplementary material, which is available to authorized users.

Background Polymorphism in antigens is a common mechanism for immune evasion used by many important pathogens, and presents major challenges in vaccine development. In malaria, many key immune targets and vaccine candidates show substantial polymorphism. However, knowledge on antigenic diversity of key antigens, the impact of polymorphism on potential vaccine escape, and how sequence polymorphism relates to antigenic differences is very limited, yet crucial for vaccine development. Plasmodium falciparum apical membrane antigen 1 (AMA1) is an important target of naturally-acquired antibodies in malaria immunity and a leading vaccine candidate. However, AMA1 has extensive allelic diversity with more than 60 polymorphic amino acid residues and more than 200 haplotypes in a single population. Therefore, AMA1 serves as an excellent model to assess antigenic diversity in malaria vaccine antigens and the feasibility of multi-allele vaccine approaches. While most previous research has focused on sequence diversity and antibody responses in laboratory animals, little has been done on the cross-reactivity of human antibodies. Methods We aimed to determine the extent of antigenic diversity of AMA1, defined by reactivity with human antibodies, and to aid the identification of specific alleles for potential inclusion in a multi-allele vaccine. We developed an approach using a multiple-antigen-competition enzyme-linked immunosorbent assay (ELISA) to examine cross-reactivity of naturally-acquired antibodies in Papua New Guinea and Kenya, and related this to differences in AMA1 sequence. Results We found that adults had greater cross-reactivity of antibodies than children, although the patterns of cross-reactivity to alleles were the same. Patterns of antibody cross-reactivity were very similar between populations (Papua New Guinea and Kenya), and over time. Further, our results show that antigenic diversity of AMA1 alleles is surprisingly restricted, despite extensive sequence polymorphism. Our findings suggest that a combination of three different alleles, if selected appropriately, may be sufficient to cover the majority of antigenic diversity in polymorphic AMA1 antigens. Antigenic properties were not strongly related to existing haplotype groupings based on sequence analysis. Conclusions Antigenic diversity of AMA1 is limited and a vaccine including a small number of alleles might be sufficient for coverage against naturally-circulating strains, supporting a multi-allele approach for developing polymorphic antigens as malaria vaccines. Electronic supplementary material The online version of this article (doi:10.1186/s12916-014-0183-5) contains supplementary material, which is available to authorized users.

Background: Platinum etoposide doublet chemotherapy remains the standard of care for SCLC; newer combination regimens have failed to improve outcome. A subgroup of patients with apparently better long-term survival present with autoimmune paraneoplastic syndromes. These reflect cross reactivity of anti-tumour immune responses with self-antigens, most frequently neuronal antigens, and suggest that SCLC is susceptible to immune control. Our study examines for the first time, whether addition of the immune stimulating anti-CTLA4 antibody Ipilimumab to carboplatin/etoposide chemotherapy can increase anti-tumour activity and whether this might come at the cost of extra immune related neurological toxicity. Methods: This phase II study examines the efficacy and toxicity of Ipilimumab (10 mg/kg) together with Carboplatin (AUC=6) and Etoposide (120 mg/m2 IV day1, 100mg BD PO days 2 and 3) chemotherapy in previously untreated patients with extensive stage (ES) SCLC. Ipilimumab was introduced in responding patients at cycles 3-6 with planned 3-monthly maintenance Ipilimumab. Primary end point is progression free survival at 1 year; secondary endpoints include 1 year overall survival and Immune related progression free survival (irPFS). Results: 39 of a planned 40 patients have entered the study with a median age of 58 yrs (range 47-84); 64% are male. Patients received at least one cycle of Ipilimumab. In 61.1% [95% CI: 44.9-75..2] >grade 3 adverse events were seen, assessed by the investigators as possibly ipilimumab related in 41.7% [95% CI: 27.1, 57.8]. Two patients (6%) had ipilimumab related neurological events, of these one was fatal, presenting like an anti-Hu syndrome. Both events occurred in patients with positive baseline anti-neuronal antibodies. Conclusions: Our multi centre study has demonstrates for the first time that chemo-immunotherapy with ICE is deliverable in ES SCLC, but with an appreciable burden of immune related toxicity. Two significant neurological immune mediated events were seen, one fatal. The data reinforce the view that SCLC is immunogenic and that immunological visibility of the tumour can link to anti neuronal immune events. Disclosure: C. Ottensmeier: Funding for this study was by an educational grant from BMS and Cancer Research UK. The trial uses the BMS proprietary compound Ipilimumab; J. Cave: Conference attendance support from BMS; M. Wheater: Conference attendance support from BMS; C. Mulatero: I have undertaken consultancy/advisory work for BMS (ipilimumab) and for Pfizer (although not about tremelimumab), and have received honoraria to attend meetings from BMS; V. Potter: I have received honoraria and consultancy fees from Roche, GSK and Lilly, but nothing from BMS; S. Danson: I sat on an advisory board and received travel expenses for a meeting from BMS - this was about 4 years ago; R. Griffiths: I have received honoraria and consultancy fees from Pfizer, Pierre-Fabre, GSK and Novartis, but nil from BMS. All other authors have declared no conflicts of interest.

4560 Background: Cure is possible for patients with relapsed germ cell tumours using further intensive cisplatin based combination chemotherapy. The Medical Research Council TIP trial reported a co...

Background: In a previous Phase 1/2b malaria vaccine trial testing the 3D7 isoform of the malaria vaccine candidate Merozoite surface protein 2 (MSP2), parasite densities in children were reduced by 62%. However, breakthrough parasitemias were disproportionately of the alternate dimorphic form of MSP2, the FC27 genotype. We therefore undertook a dose-escalating, double-blinded, placebo-controlled Phase 1 trial in healthy, malaria-naive adults of MSP2-C1, a vaccine containing recombinant forms of the two families of msp2 alleles, 3D7 and FC27 (EcMSP2-3D7 and EcMSP2-FC27), formulated in equal amounts with Montanide® ISA 720 as a water-in-oil emulsion. Methodology/Principal Findings: The trial was designed to include three dose cohorts (10, 40, and 80 μg), each with twelve subjects receiving the vaccine and three control subjects receiving Montanide® ISA 720 adjuvant emulsion alone, in a schedule of three doses at 12-week intervals. Due to unexpected local reactogenicity and concern regarding vaccine stability, the trial was terminated after the second immunisation of the cohort receiving the 40 μg dose; no subjects received the 80 μg dose. Immunization induced significant IgG responses to both isoforms of MSP2 in the 10 μg and 40 μg dose cohorts, with antibody levels by ELISA higher in the 40 μg cohort. Vaccine-induced antibodies recognised native protein by Western blots of parasite protein extracts and by immunofluorescence microscopy. Although the induced anti-MSP2 antibodies did not directly inhibit parasite growth in vitro, IgG from the majority of individuals tested caused significant antibody-dependent cellular inhibition (ADCI) of parasite growth. Conclusions/Significance: As the majority of subjects vaccinated with MSP2-C1 developed an antibody responses to both forms of MSP2, and that these antibodies mediated ADCI provide further support for MSP2 as a malaria vaccine candidate. However, in view of the reactogenicity of this formulation, further clinical development of MSP2-C1 will require formulation of MSP2 in an alternative adjuvant. Trial Registration: Australian New Zealand Clinical Trials Registry 12607000552482.

Background The polymorphic nature of many malaria vaccine candidates presents major challenges to achieving highly efficacious vaccines. Presently, there is very little knowledge on the prevalence and patterns of functional immune responses to polymorphic vaccine candidates in populations to guide vaccine design. A leading polymorphic vaccine candidate against blood-stage Plasmodium falciparum is apical membrane antigen 1 (AMA1), which is essential for erythrocyte invasion. The importance of AMA1 as a target of acquired human inhibitory antibodies, their allele specificity and prevalence in populations is unknown, but crucial for vaccine design. Methods P. falciparum lines expressing different AMA1 alleles were genetically engineered and used to quantify functional antibodies from two malaria-exposed populations of adults and children. The acquisition of AMA1 antibodies was also detected using enzyme-linked immunosorbent assay (ELISA) and competition ELISA (using different AMA1 alleles) from the same populations. Results We found that AMA1 was a major target of naturally acquired invasion-inhibitory antibodies that were highly prevalent in malaria-endemic populations and showed a high degree of allele specificity. Significantly, the prevalence of inhibitory antibodies to different alleles varied substantially within populations and between geographic locations. Inhibitory antibodies to three specific alleles were highly prevalent (FVO and W2mef in Papua New Guinea; FVO and XIE in Kenya), identifying them for potential vaccine inclusion. Measurement of antibodies by standard or competition ELISA was not strongly predictive of allele-specific inhibitory antibodies. The patterns of allele-specific functional antibody responses detected with our novel assays may indicate that acquired immunity is elicited towards serotypes that are prevalent in each geographic location. Conclusions These findings provide new insights into the nature and acquisition of functional immunity to a polymorphic vaccine candidate and strategies to quantify functional immunity in populations to guide rational vaccine design. Electronic supplementary material The online version of this article (doi:10.1186/s12916-016-0691-6) contains supplementary material, which is available to authorized users.