Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5.

There is an urgent need for improved malaria vaccine immunogens. Invasion of erythrocytes by Plasmodium falciparum is essential for its life cycle, preceding symptoms of disease and parasite transmission. Antibodies which target PfRH5 are highly effective at preventing erythrocyte invasion and the most potent growth-inhibitory antibodies bind a single epitope. Here we use structure-guided approaches to design a small synthetic immunogen, RH5-34EM which recapitulates this epitope. Structural biology and biophysics demonstrate that RH5-34EM is correctly folded and binds neutralising monoclonal antibodies with nanomolar affinity. In immunised rats, RH5-34EM induces PfRH5-targeting antibodies that inhibit parasite growth. While PfRH5-specific antibodies were induced at a lower concentration by RH5-34EM than by PfRH5, RH5-34EM induced antibodies that were a thousand-fold more growth-inhibitory as a factor of PfRH5-specific antibody concentration. Finally, we show that priming with RH5-34EM and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens.

Whole genome sequence analysis of population structure and insecticide resistance markers in Anopheles melas from the Bijagós Archipelago, Guinea-Bissau.

BACKGROUND: Anopheles melas is an understudied malaria vector with a potential role in malaria transmission on the Bijagós Archipelago of Guinea-Bissau. This study presents the first whole-genome sequencing and population genetic analysis for this species from the Bijagós. To our knowledge, this also represents the largest population genetic analysis using WGS data from non-pooled An. melas mosquitoes. METHODS: WGS was conducted for 30 individual An. melas collected during the peak malaria transmission season in 2019 from six different islands on the Bijagós Archipelago. Bioinformatics tools were used to investigate the population structure and prevalence of insecticide resistance markers in this mosquito population. RESULTS: Insecticide resistance mutations associated with pyrethroid resistance in Anopheles gambiae s.s. from the Bijagós were absent in the An. melas population, and no signatures of selective sweeps were identified in insecticide resistance-associated genes. Analysis of structural variants identified a large duplication encompassing the cytochrome-P450 gene cyp9k1. Phylogenetic analysis using publicly available mitochondrial genomes indicated that An. melas from the Bijagós split into two phylogenetic groups because of differentiation on the mitochondrial genome attributed to the cytochrome C oxidase subunits COX I and COX II and the NADH dehydrogenase subunits 1, 4, 4L and 5. CONCLUSIONS: This study identified an absence of insecticide-resistant SNPs common to An. gambiae in the An. melas population, but did identify structural variation over insecticide resistance-associated genes. Furthermore, this study presents novel insights into the population structure of this malaria vector using WGS analysis. Additional studies are required to further understand the role of this vector in malaria transmission.

A Pan Plasmodium lateral flow recombinase polymerase amplification assay for monitoring malaria parasites in vectors and human populations.

Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA-LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10-100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

Natural malaria infection elicits rare but potent neutralizing antibodies to the blood-stage antigen RH5.

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is the most advanced blood-stage malaria vaccine candidate and is being evaluated for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection, which could augment or counteract responses to vaccination. Here, we found that RH5-reactive B cells were rare, and circulating immunoglobulin G (IgG) responses to RH5 were short-lived in malaria-exposed Malian individuals, despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from eight malaria-exposed individuals mostly targeted non-neutralizing epitopes, in contrast to antibodies isolated from five RH5-vaccinated, malaria-naive UK individuals. However, MAD8-151 and MAD8-502, isolated from two malaria-exposed Malian individuals, were among the most potent neutralizers out of 186 antibodies from both cohorts and targeted the same epitopes as the most potent vaccine-induced antibodies. These results suggest that natural malaria infection may boost RH5-vaccine-induced responses and provide a clear strategy for the development of next-generation RH5 vaccines.

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex.

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

Disillusionment amongst clinical staff experiencing repeated change.

OBJECTIVE: To describe disillusionment amongst the clinical community as a result of repeated ideological and organisational change and to suggest a road map forward. CONCLUSION: Despite knowing that change can be disruptive, it will likely remain a constant and necessary feature of organisational life. Various approaches, including the development of a personal sphere of influence and knowing when to resign, are considered.

Structural basis for IL-33 recognition and its antagonism by the helminth effector protein HpARI2.

IL-33 plays a significant role in inflammation, allergy, and host defence against parasitic helminths. The model gastrointestinal nematode Heligmosomoides polygyrus bakeri secretes the Alarmin Release Inhibitor HpARI2, an effector protein that suppresses protective immune responses and asthma in its host by inhibiting IL-33 signalling. Here we reveal the structure of HpARI2 bound to mouse IL-33. HpARI2 contains three CCP-like domains, and we show that it contacts IL-33 primarily through the second and third of these. A large loop which emerges from CCP3 directly contacts IL-33 and structural comparison shows that this overlaps with the binding site on IL-33 for its receptor, ST2, preventing formation of a signalling complex. Truncations of HpARI2 which lack the large loop from CCP3 are not able to block IL-33-mediated signalling in a cell-based assay and in an in vivo female mouse model of asthma. This shows that direct competition between HpARI2 and ST2 is responsible for suppression of IL-33-dependent responses.

A Grounded Theory Exploration of Addictions Treatment Within a Commercial Airline Setting.

INTRODUCTION: An Australasian Airline's Alcohol and Other Drug (AOD) Program demonstrates abstinence rates that exceed those of general AOD programs. The reasons for this are unclear. The purpose of this research was to develop a theory as to why this program is successful.METHODS: A qualitative examination following grounded theory methodology was undertaken. AOD program patients and healthcare professionals were interviewed until content saturation was reached. Data analysis followed grounded theory to identify the key concepts associated with the program's success.RESULTS: The core theory that emerged highlighted the pivotal roles of a strong employee-company relationship, shared values, and a safety-focused culture in explicating the program's success. This moves beyond the "carrot and stick" model of motivation, where belonging to this organization and safety consciousness serve as powerful drivers for abstinence. Challenges and barriers highlighted some unique challenges to the program in managing the coronavirus pandemic and the difference in approach to substance use in community spaces versus safety-critical employment.DISCUSSION: This research expands the understanding of this AOD program's success in a safety-critical industry, emphasizing the elements of a working relationship that are beyond positive or negative reinforcement. Future research should work to quantify and test the generalizability of these findings.Nairn J, Bell E, Myers J, Higgins M, Johnston B, Newton-Howes G. A grounded theory exploration of addictions treatment within a commercial airline setting. Aerosp Med Hum Perform. 2024; 95(6):313-320.

Molecular mechanism of complement inhibition by the trypanosome receptor ISG65.

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.

Silent reflections remain unheard.

OBJECTIVE: To consider the contribution of non-clinical factors in the rising rate of mental health presentations and explore the associated silence within the psychiatric profession. CONCLUSION: Medicalisation, concept creep and group think, alongside societal demand and expectations, have collectively contributed toward a distorted view of mental health and illness. Equitable service provision has been hindered by the silence of important perspectives.

New reference genomes to distinguish the sympatric malaria parasites, Plasmodium ovale curtisi and Plasmodium ovale wallikeri.

Despite Plasmodium ovale curtisi (Poc) and wallikeri (Pow) being important human-infecting malaria parasites that are widespread across Africa and Asia, little is known about their genome diversity. Morphologically identical, Poc and Pow are indistinguishable and commonly misidentified. Recent rises in the incidence of Poc/Pow infections have renewed efforts to address fundamental knowledge gaps in their biology, and to develop diagnostic tools to understand their epidemiological dynamics and malaria burden. A major roadblock has been the incompleteness of available reference assemblies (PocGH01, PowCR01; ~ 33.5 Mbp). Here, we applied multiple sequencing platforms and advanced bioinformatics tools to generate new reference genomes, Poc221 (South Sudan; 36.0 Mbp) and Pow222 (Nigeria; 34.3 Mbp), with improved nuclear genome contiguity (> 4.2 Mbp), annotation and completeness (> 99% Plasmodium spp., single copy orthologs). Subsequent sequencing of 6 Poc and 15 Pow isolates from Africa revealed a total of 22,517 and 43,855 high-quality core genome SNPs, respectively. Genome-wide levels of nucleotide diversity were determined to be 2.98 × 10-4 (Poc) and 3.43 × 10-4 (Pow), comparable to estimates for other Plasmodium species. Overall, the new reference genomes provide a robust foundation for dissecting the biology of Poc/Pow, their population structure and evolution, and will contribute to uncovering the recombination barrier separating these species.

IL-33-binding HpARI family homologues with divergent effects in suppressing or enhancing type 2 immune responses.

HpARI is an immunomodulatory protein secreted by the intestinal nematode Heligmosomoides polygyrus bakeri, which binds and blocks IL-33. Here, we find that the H. polygyrus bakeri genome contains three HpARI family members and that these have different effects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing and HpARI3 amplifying these responses. All HpARIs have sub-nanomolar affinity for mouse IL-33; however, HpARI3 does not block IL-33-ST2 interactions. Instead, HpARI3 stabilizes IL-33, increasing the half-life of the cytokine and amplifying responses to it in vivo. Together, these data show that H. polygyrus bakeri secretes a family of HpARI proteins with both overlapping and distinct functions, comprising a complex immunomodulatory arsenal of host-targeted proteins.

Long COVID - Can we deny a diagnosis without denying a person's reality?

OBJECTIVE: The aim is to consider Long COVID not as a new clinical entity but as another example of a disabling, historical phenomenon. CONCLUSIONS: A triad of polymorphic symptomatology, an elusive pathophysiological explanation and a hostile defensiveness has appeared throughout history. The reluctance to consider these contextually may delay early intervention and appropriate patient care.

The PfRCR complex bridges malaria parasite and erythrocyte during invasion.

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

Dried Blood Spots (DBS): A suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era.

BACKGROUND: Serum or whole blood collection, processing, transport and storage still present significant challenges in low resource settings where mass surveillance is required to sustain disease elimination. Therefore, in this study, we explored the diagnostic efficacy of dried blood spots (DBS) as a minimally invasive and potentially cost-effective alternative sampling technique to whole blood sampling procedures for subsequent detection of Leishmania donovani antibodies or DNA. METHODOLOGY AND PRINCIPAL FINDINGS: Archived serum, DNA samples from whole blood of visceral leishmaniasis (VL) cases and healthy controls, and DBS from corresponding cases and controls, were used. Both molecular and serological assays were optimized to detect L. donovani antibodies or DNA in DBS elute and results were compared against those obtained with whole blood. Serological assays (both rK28 ELISA and rK39 ELISA) of DBS samples showed sensitivity and specificity of 100% and had excellent agreement with results from whole blood samples (kappa value ranged from 0.98-1). Bland-Altman analysis of OD values from rK28-ELISA with DBS elute and patients' serum showed an excellent agreement (ICC = 0.9) whereas a good agreement (ICC = 0.8) was observed in the case of rK39-ELISA. However, qPCR and RPA of DBS samples had a diminished sensitivity of 76% and 68%, respectively, and poor agreement was observed with the whole blood samples. CONCLUSION: Our results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures.

Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes.

Basigin is an essential host receptor for invasion of Plasmodium falciparum into human erythrocytes, interacting with parasite surface protein PfRH5. PfRH5 is a leading blood-stage malaria vaccine candidate and a target of growth-inhibitory antibodies. Here, we show that erythrocyte basigin is exclusively found in one of two macromolecular complexes, bound either to plasma membrane Ca2+-ATPase 1/4 (PMCA1/4) or to monocarboxylate transporter 1 (MCT1). PfRH5 binds to each of these complexes with a higher affinity than to isolated basigin ectodomain, making it likely that these are the physiological targets of PfRH5. PMCA-mediated Ca2+ export is not affected by PfRH5, making it unlikely that this is the mechanism underlying changes in calcium flux at the interface between an erythrocyte and the invading parasite. However, our studies rationalise the function of the most effective growth-inhibitory antibodies targeting PfRH5. While these antibodies do not reduce the binding of PfRH5 to monomeric basigin, they do reduce its binding to basigin-PMCA and basigin-MCT complexes. This indicates that the most effective PfRH5-targeting antibodies inhibit growth by sterically blocking the essential interaction of PfRH5 with basigin in its physiological context.

Trypanosomes and complement: more than one way to die?

African trypanosomes show a remarkable ability to survive as extracellular parasites in the blood and tissue spaces of an infected mammal. Throughout the infection they are exposed to the molecules and cells of the immune system, including complement. In this opinion piece, we review decades-worth of evidence about how complement affects African trypanosomes. We highlight the discovery of a trypanosome receptor for complement C3 and we critically assess three recent studies which attempt to provide a structural and mechanistic view of how this receptor helps trypanosomes to survive in the presence of complement.

Outcomes of an Integrated Multidisciplinary Clinic for People with Functional Neurological Disorder.

Background: Functional neurological disorder (FND) is a disabling condition which has poor prognosis without treatment. This study aimed to evaluate the effectiveness of an outpatient integrated multidisciplinary intervention for the condition. Objectives: This study aimed to assess the outcomes of a pilot integrated multidisciplinary treatment clinic for FND with motor symptoms. Methods: Patients were seen simultaneously by a neurology doctor, a physiotherapist, a clinical psychologist, and sometimes a psychiatrist. The primary endpoint was change in quality of life measured by Short Form-36 (SF-36). Secondary outcomes were change in work and social participation measured by the Work and Social Adjustment Scale (WSAS), ability to participate in full-time or part-time employment, self-rated understanding of FND, and self-rated agreement with the diagnosis of FND. Over the year, 13 patients were recruited to the clinic, and 11 agreed to participate in the outcome study. Results: Statistically significant improvements in quality of life were seen across seven out of eight domains of the SF-36, with improvements in individual domains of between 23 and 39 points (of a possible 100). Mean Work and Social Adjustment Scale score halved from 26 to 13 (worst possible is 40). Of the 12 patients treated, one began to work again after complete unemployment, and two who had been working reduced hours due to disability resumed full time work. No patients had worsened occupational status. Conclusions: This intervention is associated with substantial improvements in quality of life and function, and may be more amenable to delivery at non-specialist centers than other described interventions for FND.

Structural basis for DARC binding in reticulocyte invasion by Plasmodium vivax.

The symptoms of malaria occur during the blood stage of infection, when the parasite replicates within human red blood cells. The human malaria parasite, Plasmodium vivax, selectively invades reticulocytes in a process which requires an interaction between the ectodomain of the human DARC receptor and the Plasmodium vivax Duffy-binding protein, PvDBP. Previous studies have revealed that a small helical peptide from DARC binds to region II of PvDBP (PvDBP-RII). However, it is also known that sulphation of tyrosine residues on DARC affects its binding to PvDBP and these residues were not observed in previous structures. We therefore present the structure of PvDBP-RII bound to sulphated DARC peptide, showing that a sulphate on tyrosine 41 binds to a charged pocket on PvDBP-RII. We use molecular dynamics simulations, affinity measurements and growth-inhibition experiments in parasites to confirm the importance of this interaction. We also reveal the epitope for vaccine-elicited growth-inhibitory antibody DB1. This provides a complete understanding of the binding of PvDBP-RII to DARC and will guide the design of vaccines and therapeutics to target this essential interaction.

Accelerated CMC workflows to enable speed to clinic in the COVID-19 era: A multi-company view from the biopharmaceutical industry.

The COVID-19 pandemic has placed unprecedented pressure on biopharmaceutical companies to develop efficacious preventative and therapeutic treatments, which is unlikely to abate in the coming years. The importance of fast progress to clinical evaluation for treatments, which tackle unmet medical needs puts strain on traditional product development timelines, which can take years from start to finish. Although previous work has been successful in reducing phase 1 timelines for recombinant antibodies, through utilization of the latest technological advances and acceptance of greater business risk or costs, substantially faster development is likely achievable without increased risk to patients during initial clinical evaluation. To optimize lessons learned from the pandemic and maximize multi-stakeholder (i.e., patients, clinicians, companies, regulatory agencies) benefit, we conducted an industry wide benchmarking survey in September/October 2021. The aims of this survey were to: (i) benchmark current technical practices of key process and product development activities related to manufacturing of therapeutic proteins, (ii) understand the impact of changes implemented in COVID-19 accelerated Ab programs, and whether any such changes can be retained as part of sustainable long-term business practices and (iii) understand whether any accelerative action(s) taken have (negatively) impacted the wider development process. This article provides an in-depth analysis of this data, ultimately highlighting an industry perspective of how biopharmaceutical companies can sustainably adopt new approaches to therapeutic protein development and production.