Correction: The physicochemical fingerprint of Necator americanus.

[This corrects the article DOI: 10.1371/journal.pntd.0005971.].

Controlled Infection of Humans with the Hookworm Parasite Necator americanus to Accelerate Vaccine Development : The Human Hookworm Vaccination/Challenge Model (HVCM).

In this chapter, we describe the scientific, technical, clinical and regulatory aspects of establishing a controlled human hookworm infection (CHHI) model in non-endemic and endemic geographical regions, to facilitate a pathway towards accelerated vaccine development. The success achieved in establishing the CHHI platform specifically allows the Human Hookworm Vaccine Initiative (HHVI) to accelerate its progress by establishing a human hookworm vaccination/challenge model (HVCM) in a hookworm endemic area of Brazil. The HVCM will permit the rapid and robust determination of clinical efficacy in adults, allowing for early selection of the most efficacious human hookworm vaccine (HHV) candidate(s) to advance into later-stage pivotal paediatric clinical trials and reduce the overall number of participants required to assess efficacy (Diemert et al. 2018).

Experimental infection with the hookworm, Necator americanus, is associated with stable gut microbial diversity in human volunteers with relapsing multiple sclerosis.

BACKGROUND: Helminth-associated changes in gut microbiota composition have been hypothesised to contribute to the immune-suppressive properties of parasitic worms. Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system whose pathophysiology has been linked to imbalances in gut microbial communities. RESULTS: In the present study, we investigated, for the first time, qualitative and quantitative changes in the faecal bacterial composition of human volunteers with remitting multiple sclerosis (RMS) prior to and following experimental infection with the human hookworm, Necator americanus (N+), and following anthelmintic treatment, and compared the findings with data obtained from a cohort of RMS patients subjected to placebo treatment (PBO). Bacterial 16S rRNA high-throughput sequencing data revealed significantly decreased alpha diversity in the faecal microbiota of PBO compared to N+ subjects over the course of the trial; additionally, we observed significant differences in the abundances of several bacterial taxa with putative immune-modulatory functions between study cohorts. Parabacteroides were significantly expanded in the faecal microbiota of N+ individuals for which no clinical and/or radiological relapses were recorded at the end of the trial. CONCLUSIONS: Overall, our data lend support to the hypothesis of a contributory role of parasite-associated alterations in gut microbial composition to the immune-modulatory properties of hookworm parasites.

The evolution of IgE-mediated type I hypersensitivity and its immunological value.

The allergic phenotype manifests itself in a spectrum of troublesome to life-threatening diseases, from seasonal hay fever, through the food allergies, atopic eczema, asthma, to anaphylaxis. Allergy, that is an overreaction to allergen in hypersensitive individuals, results from the production of IgE, mast cell and basophil sensitisation and degranulation, requiring a range of medications to manage the conditions. Yet it is highly likely that allergy evolved for a purpose and that allergic diseases are accidental consequences of an insufficiently regulated immune response. This article presents a viewpoint from which to restore the immunological reputation of the allergic phenotype. We consider the evolutionary origins of potential allergens, toxins and parasites, and how they might have influenced early-mammal species in existence when IgE first developed. We conclude that the allergic phenotype has likely saved the lives of many more mammals than have ever died from allergy, so justifying the positive role of IgE in our evolution.

An Absence of Epstein-Barr Virus Reactivation and Associations with Disease Activity in People with Multiple Sclerosis Undergoing Therapeutic Hookworm Vaccination.

Background: Epstein-Barr virus (EBV) infection is strongly associated with multiple sclerosis (MS). Helminth infection can downregulate antiviral immune responses, potentially protecting against MS, but with a theoretical risk for reactivating latent EBV infection. Objective: To investigate parameters of EBV infection and their relationship with disease activity in people with MS (PwMS) therapeutically vaccinated with Necator americanus (hookworm). Methods: Sequential serum samples from 51 PwMS; 26 therapeutically infected (25 larvae) with N. americanus and 25 controls were tested for EBV virus capsid antigen (VCA) IgG and IgM, EBV nuclear antigen-1 (EBNA-1) IgG, and EBV early antigen (EA) IgG. Disease activity was assessed by periodic MRI. Significance was set at p < 0.05. Results: All PwMS were EBV VCA IgG and EBNA-1 IgG positive, and 35.2% were EBV EA IgG positive. EBV antibody levels were generally stable, and EBV reactivation in PwMS was not demonstrated by significant increases in IgG titre over 12 months. Disease activity was most frequent in PwMS possessing high levels of EBV VCA IgG (>600 units/mL) or EBNA-1 IgG (>150 units/mL); however, there was no association with hookworm treatment. Interpretation: Therapeutic hookworm vaccination was not associated with EBV reactivation. Multiple sclerosis disease activity was associated with high levels of EBV VCA IgG or EBNA-1 IgG.

Hookworm Treatment for Relapsing Multiple Sclerosis: A Randomized Double-Blinded Placebo-Controlled Trial.

Importance: Studies suggest gut worms induce immune responses that can protect against multiple sclerosis (MS). To our knowledge, there are no controlled treatment trials with helminth in MS. Objective: To determine whether hookworm treatment has effects on magnetic resonance imaging (MRI) activity and T regulatory cells in relapsing MS. Design, Setting, and Participants: This 9-month double-blind, randomized, placebo-controlled trial was conducted between September 2012 and March 2016 in a modified intention-to-treat population (the data were analyzed June 2018) at the University of Nottingham, Queen's Medical Centre, a single tertiary referral center. Patients aged 18 to 61 years with relapsing MS without disease-modifying treatment were recruited from the MS clinic. Seventy-three patients were screened; of these, 71 were recruited (2 ineligible/declined). Interventions: Patients were randomized (1:1) to receive either 25 Necator americanus larvae transcutaneously or placebo. The MRI scans were performed monthly during months 3 to 9 and 3 months posttreatment. Main Outcomes and Measures: The primary end point was the cumulative number of new/enlarging T2/new enhancing T1 lesions at month 9. The secondary end point was the percentage of cluster of differentiation (CD) 4+CD25highCD127negT regulatory cells in peripheral blood. Results: Patients (mean [SD] age, 45 [9.5] years; 50 women [71%]) were randomized to receive hookworm (35 [49.3%]) or placebo (36 [50.7%]). Sixty-six patients (93.0%) completed the trial. The median cumulative numbers of new/enlarging/enhancing lesions were not significantly different between the groups by preplanned Mann-Whitney U tests, which lose power with tied data (high number of zeroactivity MRIs in the hookworm group, 18/35 [51.4%] vs 10/36 [27.8%] in the placebo group). The percentage of CD4+CD25highCD127negT cells increased at month 9 in the hookworm group (hookworm, 32 [4.4%]; placebo, 34 [3.9%]; P = .01). No patients withdrew because of adverse effects. There were no differences in adverse events between groups except more application-site skin discomfort in the hookworm group (82% vs 28%). There were 5 relapses (14.3%) in the hookworm group vs 11 (30.6%) receiving placebo. Conclusions and Relevance: Treatment with hookworm was safe and well tolerated. The primary outcome did not reach significance, likely because of a low level of disease activity. Hookworm infection increased T regulatory cells, suggesting an immunobiological effect of hookworm. It appears that a living organism can precipitate immunoregulatory changes that may affect MS disease activity. Trial Registration: ClinicalTrials.gov Identifier: NCT01470521.

What is the optimal treatment time for larval therapy? A study on incubation time and tissue debridement by bagged maggots of the greenbottle fly, Lucilia sericata.

The effective use of larvae of the greenbottle fly, Lucilia sericata, in wound debridement requires a working knowledge of how feeding changes over time. Using a laboratory assay and bagged larval dressings, the effect of incubation time on larval feeding rates and body mass was investigated for up to 120 hours at 32°C. The mass of tissue digested increased significantly in incremental 24-hour periods up to 72 hours, with no significant consumption occurring afterwards. Larval mass increased only up to 48 hours. A further test comparing the efficacy of a single 96-hour application of larvae against two consecutive 48-hour applications found that the mass of tissue digested in the latter was 14.3% higher than the former, a difference that was statistically significant. Current clinical guidance suggests a 4-day application period for bagged larvae. Based on these results, an incubation time of 72 hours (3 days) for bagged larvae would be the most effective at the study temperature. However, it is acknowledged that wound temperature can vary, whereby feeding rates would likely differ. In view of this, we conclude that a period of 3 to 4 days is optimum for the application of larvae, and current guidelines should be adhered to.

Haematophagic Caenorhabditis elegans.

Caenorhabditis elegans is a free-living nematode that resides in soil and typically feeds on bacteria. We postulate that haematophagic C. elegans could provide a model to evaluate vaccine responses to intestinal proteins from hematophagous nematode parasites, such as Necator americanus. Human erythrocytes, fluorescently labelled with tetramethylrhodamine succinimidyl ester, demonstrated a stable bright emission and facilitated visualization of feeding events with fluorescent microscopy. C. elegans were observed feeding on erythrocytes and were shown to rupture red blood cells upon capture to release and ingest their contents. In addition, C. elegans survived equally on a diet of erythrocytes. There was no statistically significant difference in survival when compared with a diet of Escherichia coli OP50. The enzymes responsible for the digestion and detoxification of haem and haemoglobin, which are key components of the hookworm vaccine, were found in the C. elegans intestine. These findings support our postulate that free-living nematodes could provide a model for the assessment of neutralizing antibodies to current and future hematophagous parasite vaccine candidates.

Controlled Human Hookworm Infection: Accelerating Human Hookworm Vaccine Development.

BACKGROUND: Controlled human hookworm infection (CHHI) is a central component of a proposed hookworm vaccination-challenge model (HVCM) to test the efficacy of candidate vaccines. Critical to CHHI is the manufacture of Necator americanus infective larvae (NaL3) according to current Good Manufacturing Practice (cGMP) and the determination of an inoculum of NaL3 that is safe and reliably induces patent infection. METHODS: cGMP-grade NaL3 were produced for a phase 1 trial in 20 healthy, hookworm-naïve adults in the United States, who received either 25 or 50 NaL3. Participants were monitored for 12-18 weeks postinfection for safety, tolerability, and patency of N. americanus infection. RESULTS: Both NaL3 doses were well tolerated. Early manifestations of infection included pruritus, pain, and papulovesicular rash at the application site. Gastrointestinal symptoms and eosinophilia appeared after week 4 postinfection. The 50 NaL3 inoculum induced patent N. americanus infection in 90% of this dose group. CONCLUSIONS: The inoculum of 50 NaL3 was well tolerated and consistently induced patent N. americanus infection suitable for future HVCM trials. CLINICAL TRIALS REGISTRATION: NCT01940757.

The physicochemical fingerprint of Necator americanus.

Necator americanus, a haematophagous hookworm parasite, infects ~10% of the world's population and is considered to be a significant public health risk. Its lifecycle has distinct stages, permitting its successful transit from the skin via the lungs (L3) to the intestinal tract (L4 maturing to adult). It has been hypothesised that the L3 larval sheath, which is shed during percutaneous infection (exsheathment), diverts the immune system to allow successful infection and reinfection in endemic areas. However, the physicochemical properties of the L3 larval cuticle and sheath, which are in direct contact with the skin and its immune defences, are unknown. In the present study, we controlled exsheathment, to characterise the sheath and underlying cuticle surfaces in situ, using atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). AFM revealed previously unseen surface area enhancing nano-annuli exclusive to the sheath surface and confirmed greater adhesion forces exist between cationic surfaces and the sheath, when compared to the emergent L3 cuticle. Furthermore, ToF-SIMS elucidated different chemistries between the surfaces of the cuticle and sheath which could be of biological significance. For example, the phosphatidylglycerol rich cuticle surface may support the onward migration of a lubricated infective stage, while the anionic and potentially immunologically active heparan sulphate rich deposited sheath could result in the diversion of immune defences to an inanimate antigenic nidus. We propose that our initial studies into the surface analysis of this hookworm provides a timely insight into the physicochemical properties of a globally important human pathogen at its infective stage and anticipate that the development and application of this analytical methodology will support translation of these findings into a biological context.

Endotoxin testing of a wound debridement device containing medicinal Lucilia sericata larvae.

Alimentary products of medicinal Lucilia sericata larvae are studied to determine their mechanisms of action, particularly in the contexts of wound debridement and disinfection. Furthermore, the larvae can be applied to patients in contained medical devices (such as the BioBag; BioMonde). Here, we tested the materials and larval content of the most commonly used debridement device (the "BB-50") to explore the possibility that endotoxins may be contributing to the bio-activity of the product, given that endotoxins are potent stimulants of cellular activation. Using standardised protocols to collect larval alimentary products (LAP), we proceeded to determine residual endotoxin levels in LAP derived from the device, before and after the neutralisation of interfering enzymatic activity. The debridement device and its associated larval content was not a significant source of lipopolysaccharide (LPS) activity. However, it is clear from these experiments that a failure to remove the confounding serine proteinase activity would have resulted in spuriously high and erroneous results. The residual LPS levels detected are unlikely to be active in wound healing assays, following cross-referencing to publications where LPS at much higher levels has been shown to have positive and negative effects on processes associated with wound repair and tissue regeneration.

TIME management by medicinal larvae.

Wound bed preparation (WBP) is an integral part of the care programme for chronic wounds. The acronym TIME is used in the context of WBP and describes four barriers to healing in chronic wounds; namely, dead Tissue, Infection and inflammation, Moisture imbalance and a non-migrating Edge. Larval debridement therapy (LDT) stems from observations that larvae of the blowfly Lucilia sericata clean wounds of debris. Subsequent clinical studies have proven debriding efficacy, which is likely to occur as a result of enzymatically active alimentary products released by the insect. The antimicrobial, anti-inflammatory and wound healing activities of LDT have also been investigated, predominantly in a pre-clinical context. This review summarises the findings of investigations into the molecular mechanisms of LDT and places these in context with the clinical concept of WBP and TIME. It is clear from these findings that biotherapy with L. sericata conforms with TIME, through the enzymatic removal of dead tissue and its associated biofilm, coupled with the secretion of defined antimicrobial peptides. This biotherapeutic impact on the wound serves to reduce inflammation, with an associated capacity for an indirect effect on moisture imbalance. Furthermore, larval serine proteinases have the capacity to alter fibroblast behaviour in a manner conducive to the formation of granulation tissue.

Degradation of MSCRAMM target macromolecules in VLU slough by Lucilia sericata chymotrypsin 1 (ISP) persists in the presence of tissue gelatinase activity.

Venous leg ulcer slough is unpleasant to the patient and difficult to manage clinically. It harbours infection, also preventing wound management materials and dressings from supporting the underlying viable tissues. In other words, slough has significant nuisance value in the tissue viability clinic. In this study, we have sought to increase our knowledge of slough by building upon a previous but limited analysis of this necrotic tissue. In particular, slough has been probed using Western blotting for the presence of proteins with the capacity to engage microbial surface components recognising adhesive matrix macromolecules. Although the samples were difficult to resolve, we detected fibrinogen, fibronectin, IgG, collagen, human serum albumin and matrix metalloproteinase-9. Furthermore, the effect of a maggot-derived debridement enzyme, chymotrypsin 1 on macromolecules in slough was confirmed across seven patient samples. The effect of chymotrypsin 1 on slough confirms our thesis that this potential debridement enzyme could be effective in removing slough along with its associated bacteria, given its observed resistance to intrinsic gelatinase activity. In summary, we believe that the data provide scientists and clinicians with further insights into the potential molecular interactions between bacteria, wound tissue and Lucilia sericata in a clinically problematic yet scientifically interesting wound ecosystem.

Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1.

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe (3)H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. (3)H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on (3)H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in (3)H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.

Bacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypes.

Bacteria deploy a range of chemistries to regulate their behaviour and respond to their environment. Quorum sensing is one method by which bacteria use chemical reactions to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the chemical reactions used for quorum sensing are therefore a potential means to control bacterial population responses. Here, we report how polymeric 'bacteria sequestrants', designed to bind to bacteria through electrostatic interactions and therefore inhibit bacterial adhesion to surfaces, induce the expression of quorum-sensing-controlled phenotypes as a consequence of cell clustering. A combination of polymer and analytical chemistry, biological assays and computational modelling has been used to characterize the feedback between bacteria clustering and quorum sensing signalling. We have also derived design principles and chemical strategies for controlling bacterial behaviour at the population level.

Multiple actions of Lucilia sericata larvae in hard-to-heal wounds: larval secretions contain molecules that accelerate wound healing, reduce chronic inflammation and inhibit bacterial infection.

In Europe ≈15,000 patients receive larval therapy for wound treatment annually. Over the past few years, clinical studies have demonstrated the success of larvae of Lucilia sericata as debridement agents. This is based on a combination of physical and biochemical actions. Laboratory investigations have advanced our understanding of the biochemical mechanisms underlying the beneficial effects of larval secretions, including removal of dead tissue, reduction of the bacterial burden, and promotion of tissue regeneration. The present article summarizes our current understanding of the microbiological, immunological, and wound healing actions of larval therapy, and the molecules involved in these beneficial effects. Future studies will focus on the isolation, identification, and (pre)clinical testing of the effective molecules of L. sericata larvae. These molecules may be candidates for the development of new agents for the treatment of several infectious and inflammatory diseases, including chronic wounds.

Mapping the pharyngeal and intestinal pH of Caenorhabditis elegans and real-time luminal pH oscillations using extended dynamic range pH-sensitive nanosensors.

Extended dynamic range pH-sensitive ratiometric nanosensors, capable of accurately mapping the full physiological pH range, have been developed and used to characterize the pH of the pharyngeal and intestinal lumen of Caenorhabditis elegans in real-time. Nanosensors, 40 nm in diameter, were prepared by conjugating pH-sensitive fluorophores, carboxyfluorescein (FAM) and Oregon Green (OG) in a 1:1 ratio, and a reference fluorophore, 5-(and-6)-carboxytetramethylrhodamine (TAMRA) to an inert polyacrylamide matrix. Accurate ratiometric pH measurements were calculated through determination of the fluorescence ratio between the pH-sensitive and reference fluorophores. Nanosensors were calibrated with an automated image analysis system and validated to demonstrate a pH measurement resolution of ±0.17 pH units. The motility of C. elegans populations, as an indicator for viability, showed nematodes treated with nanosensors, for concentrations ranging from 50.00 to 3.13 mg/mL, were not statistically different to nematodes not challenged with nanosensors up to a period of 4 days (p < 0.05). The nanosensors were also found to remain in the C. elegans lumen >24 h after nanosensor challenge was removed. The pH of viable C. elegans lumen was found to range from 5.96 ± 0.31 in the anterior pharynx to 3.59 ± 0.09 in the posterior intestine. The pharyngeal pumping rate, which dictates the transfer of ingested material from the pharynx to the intestine, was found to be temperature dependent. Imaging C. elegans at 4 °C reduced the pharyngeal pumping rate to 7 contractions/min and enabled the reconstruction of rhythmic pH oscillations in the intestinal lumen in real-time with fluorescence microscopy.

Lucilia sericata chymotrypsin disrupts protein adhesin-mediated staphylococcal biofilm formation.

Staphylococcus aureus and Staphylococcus epidermidis biofilms cause chronic infections due to their ability to form biofilms. The excretions/secretions of Lucilia sericata larvae (maggots) have effective activity for debridement and disruption of bacterial biofilms. In this paper, we demonstrate how chymotrypsin derived from maggot excretions/secretions disrupts protein-dependent bacterial biofilm formation mechanisms.

Expression of a cGMP compatible Lucilia sericata insect serine proteinase debridement enzyme.

Previously, we demonstrated the effectiveness of a research grade recombinant chymotrypsin, derived from the larvae of Lucilia sericata, in "debriding" slough/eschar from venous leg ulcers ex vivo. Furthermore, we were able to formulate this enzyme for successful delivery to in vitro wound healing assays, from a prototype hydrogel wound dressing, and showed that enzyme delivered in this way could degrade wound tissue ex vivo. Recently, to progress biotechnological development of the enzyme as a potential therapeutic product, we explored expression using current good manufacturing practice (cGMP) guidelines, and now report that a recombinant chymotrypsin I zymogen from L. sericata can be expressed in the cGMP acceptable strain of Escherichia coli (BLR-DE3). In addition, the conditions required for purification, refolding and activation of the chymotrypsinogen have been determined. The activated enzyme was stable, and effective in digesting wound slough/eschar tissue. To summarise, we have successfully initiated the production and characterisation of a novel cGMP compatible product for use in future clinical trials.