Talking in primary care (TIP): protocol for a cluster-randomised controlled trial in UK primary care to assess clinical and cost-effectiveness of communication skills e-learning for practitioners on patients' musculoskeletal pain and enablement.

INTRODUCTION: Effective communication can help optimise healthcare interactions and patient outcomes. However, few interventions have been tested clinically, subjected to cost-effectiveness analysis or are sufficiently brief and well-described for implementation in primary care. This paper presents the protocol for determining the effectiveness and cost-effectiveness of a rigorously developed brief eLearning tool, EMPathicO, among patients with and without musculoskeletal pain. METHODS AND ANALYSIS: A cluster randomised controlled trial in general practitioner (GP) surgeries in England and Wales serving patients from diverse geographic, socioeconomic and ethnic backgrounds. GP surgeries are randomised (1:1) to receive EMPathicO e-learning immediately, or at trial end. Eligible practitioners (eg, GPs, physiotherapists and nurse practitioners) are involved in managing primary care patients with musculoskeletal pain. Patient recruitment is managed by practice staff and researchers. Target recruitment is 840 adults with and 840 without musculoskeletal pain consulting face-to-face, by telephone or video. Patients complete web-based questionnaires at preconsultation baseline, 1 week and 1, 3 and 6 months later. There are two patient-reported primary outcomes: pain intensity and patient enablement. Cost-effectiveness is considered from the National Health Service and societal perspectives. Secondary and process measures include practitioner patterns of use of EMPathicO, practitioner-reported self-efficacy and intentions, patient-reported symptom severity, quality of life, satisfaction, perceptions of practitioner empathy and optimism, treatment expectancies, anxiety, depression and continuity of care. Purposive subsamples of patients, practitioners and practice staff take part in up to two qualitative, semistructured interviews. ETHICS APPROVAL AND DISSEMINATION: Approved by the South Central Hampshire B Research Ethics Committee on 1 July 2022 and the Health Research Authority and Health and Care Research Wales on 6 July 2022 (REC reference 22/SC/0145; IRAS project ID 312208). Results will be disseminated via peer-reviewed academic publications, conference presentations and patient and practitioner outlets. If successful, EMPathicO could quickly be made available at a low cost to primary care practices across the country. TRIAL REGISTRATION NUMBER: ISRCTN18010240.

A forward genetic screen reveals a primary role for Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a and 2b in determining alternative erythrocyte invasion pathways.

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.

Salvage Chemotherapy With Gemcitabine, Paclitaxel, Ifosfamide, and Cisplatin for Relapsed Germ Cell Cancer.

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.

An in vitro erythrocyte preference assay reveals that Plasmodium falciparum parasites prefer Type O over Type A erythrocytes.

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.

Human Immunization With a Polymorphic Malaria Vaccine Candidate Induced Antibodies to Conserved Epitopes That Promote Functional Antibodies to Multiple Parasite Strains.

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.

Synergistic malaria vaccine combinations identified by systematic antigen screening.

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.

P113 is a merozoite surface protein that binds the N terminus of Plasmodium falciparum RH5.

Invasion of erythrocytes by Plasmodium falciparum merozoites is necessary for malaria pathogenesis and is therefore a primary target for vaccine development. RH5 is a leading subunit vaccine candidate because anti-RH5 antibodies inhibit parasite growth and the interaction with its erythrocyte receptor basigin is essential for invasion. RH5 is secreted, complexes with other parasite proteins including CyRPA and RIPR, and contains a conserved N-terminal region (RH5Nt) of unknown function that is cleaved from the native protein. Here, we identify P113 as a merozoite surface protein that directly interacts with RH5Nt. Using recombinant proteins and a sensitive protein interaction assay, we establish the binding interdependencies of all the other known RH5 complex components and conclude that the RH5Nt-P113 interaction provides a releasable mechanism for anchoring RH5 to the merozoite surface. We exploit these findings to design a chemically synthesized peptide corresponding to RH5Nt, which could contribute to a cost-effective malaria vaccine.

A novel approach to identifying patterns of human invasion-inhibitory antibodies guides the design of malaria vaccines incorporating polymorphic antigens.

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.

Outcome and Biomarker Analysis from a Multicenter Phase 2 Study of Ipilimumab in Combination with Carboplatin and Etoposide as First-Line Therapy for Extensive-Stage SCLC.

OBJECTIVES: Our aim was to evaluate the safety and efficacy of ipilimumab combined with standard first-line chemotherapy for patients with extensive-stage SCLC. METHODS: Patients with chemotherapy-naive extensive-stage SCLC were treated with carboplatin and etoposide for up to six cycles. Ipilimumab, 10 mg/kg, was given on day 1 of cycles 3 to 6 and every 12 weeks. Response was assessed by the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.0, and immune-related response criteria. The primary end point was 1-year progression-free survival (PFS) according to RECIST. Secondary end points included PFS according to immune-related PFS and overall survival. Autoantibody serum levels were evaluated and correlated with clinical outcomes. RESULTS: A total of 42 patients were enrolled between September 2011 and April 2014; 39 were evaluable for safety and 38 for efficacy. Six of 38 patients (15.8% [95% confidence interval (CI): 7.4-30.4]) were alive and progression-free at 1-year by RECIST. Median PFS was 6.9 months (95% CI: 5.5-7.9). Median immune-related PFS was 7.3 months (95% CI: 5.5-8.8). Median overall survival was 17.0 months (95% CI: 7.9-24.3). Of the patients evaluable for response, 21 of 29 (72.4%) achieved an objective response by RECIST and 28 of 33 (84.8%) achieved an objective response by the immune-related response criteria. All patients experienced at least one adverse event; at least one grade 3 or higher toxicity developed in 35 of 39 patients (89.7%); in 27 patients (69.2%) this was related to ipilimumab. Five deaths were reported to be related to ipilimumab. Positivity of an autoimmune profile at baseline was associated with improved outcomes and severe neurological toxicity. CONCLUSIONS: Ipilimumab in combination with carboplatin and etoposide might benefit a subgroup of patients with advanced SCLC. Autoantibody analysis correlates with treatment benefit and toxicity and warrants further investigation.

Differences in affinity of monoclonal and naturally acquired polyclonal antibodies against Plasmodium falciparum merozoite antigens.

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.

Limited antigenic diversity of Plasmodium falciparum apical membrane antigen 1 supports the development of effective multi-allele vaccines.

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.

Identification and prioritization of merozoite antigens as targets of protective human immunity to Plasmodium falciparum malaria for vaccine and biomarker development.

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.

New insights into acquisition, boosting, and longevity of immunity to malaria in pregnant women.

BACKGROUND: How antimalarial antibodies are acquired and maintained during pregnancy and boosted after reinfection with Plasmodium falciparum and Plasmodium vivax is unknown. METHODS: A nested case-control study of 467 pregnant women (136 Plasmodium-infected cases and 331 uninfected control subjects) in northwestern Thailand was conducted. Antibody levels to P. falciparum and P. vivax merozoite antigens and the pregnancy-specific PfVAR2CSA antigen were determined at enrollment (median 10 weeks gestation) and throughout pregnancy until delivery. RESULTS: Antibodies to P. falciparum and P. vivax were highly variable over time, and maintenance of high levels of antimalarial antibodies involved highly dynamic responses resulting from intermittent exposure to infection. There was evidence of boosting with each successive infection for P. falciparum responses, suggesting the presence of immunological memory. However, the half-lives of Plasmodium antibody responses were relatively short, compared with measles (457 years), and much shorter for merozoite responses (0.8-7.6 years), compared with PfVAR2CSA responses (36-157 years). The longer half-life of antibodies to PfVAR2CSA suggests that antibodies acquired in one pregnancy may be maintained to protect subsequent pregnancies. CONCLUSIONS: These findings may have important practical implications for predicting the duration of vaccine-induced responses by candidate antigens and supports the development of malaria vaccines to protect pregnant women.

A phase 1 trial of MSP2-C1, a blood-stage malaria vaccine containing 2 isoforms of MSP2 formulated with Montanide® ISA 720.

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-naïve 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.