Rebecca Ashfield

Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1

ATP-sensitive potassium channels play a major role in linking metabolic signals to the exocytosis of insulin in the pancreatic beta cell. These channels consist of two types of protein subunit: the sulfonylurea receptor SUR1 and the inward rectifying potassium channel Kir6.2. Mutations in the genes encoding these proteins are the most common cause of congenital hyperinsulinism (CHI). Since 1973, we have followed up 38 pediatric CHI patients in Finland. We reported previously that a loss-of-function mutation in SUR1 (V187D) is responsible for CHI of the most severe cases. We have now identified a missense mutation, E1506K, within the second nucleotide binding fold of SUR1, found heterozygous in seven related patients with CHI and in their mothers. All patients have a mild form of CHI that usually can be managed by long-term diazoxide treatment. This clinical finding is in agreement with the results of heterologous coexpression studies of recombinant Kir6.2 and SUR1 carrying the E1506K mutation. Mutant K(ATP) channels were insensitive to metabolic inhibition, but a partial response to diazoxide was retained. Five of the six mothers, two of whom suffered from hypoglycemia in infancy, have developed gestational or permanent diabetes. Linkage and haplotype analysis supported a dominant pattern of inheritance in a large pedigree. In conclusion, we describe the first dominantly inherited SUR1 mutation that causes CHI in early life and predisposes to later insulin deficiency.

Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.

1. We have studied the electrophysiological properties of cloned ATP‐sensitive K+ channels (KATP channels) heterologously expressed in Xenopus oocytes. This channel comprises a sulphonylurea receptor subunit (SUR) and an inwardly rectifying K+ channel subunit (Kir). 2. Oocytes injected with SUR1 and either Kir6.2 or Kir6.1 exhibited large inwardly rectifying K+ currents when cytosolic ATP levels were lowered by the metabolic inhibitors azide or FCCP. No currents were observed in response to azide in oocytes injected with Kir6.2, Kir6.1 or SUR1 alone, indicating that both the sulphonylurea receptor (SUR1) and an inward rectifier (Kir6.1 or Kir6.2) are needed for functional channel activity. 3. The pharmacological properties of Kir6.2‐SUR1 currents resembled those of native beta‐cell ATP‐sensitive K+ channel currents (KATP currents): the currents were > 90% blocked by tolbutamide (500 microM), meglitinide (10 microM) or glibenclamide (100 nM), and activated 1.8‐fold by diazoxide (340 microM), 1.4‐fold by pinacidil (1 mM) and unaffected by cromakalim (0.5 mM). 4. Macroscopic Kir6.2‐SUR1 currents in inside‐out patches were inhibited by ATP with a Ki of 28 microM. Kir6.1‐SUR1 currents ran down within seconds of patch excision preventing analysis of ATP sensitivity. 5. No sensitivity to tolbutamide or metabolic inhibition was observed when SUR1 was coexpressed with either Kir1.1a or Kir2.1, suggesting that these proteins do not couple in Xenopus ocytes. 6. Our data demonstrate that the Xenopus oocyte constitutes a good expression system for cloned KATP channels and that expression may be assayed by azide‐induced metabolic inhibition.

Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies.

Invasion of host erythrocytes is essential to the life cycle of Plasmodium parasites and development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by coordinated release of specialized apical organelles and their parasite protein contents. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and erythrocyte-binding like proteins, which mediate host–parasite interactions. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin (also known as CD147 and EMMPRIN). Antibodies targeting PfRH5 or basigin efficiently block parasite invasion in vitro, making PfRH5 an excellent vaccine candidate. Here we present crystal structures of PfRH5 in complex with basigin and two distinct inhibitory antibodies. PfRH5 adopts a novel fold in which two three-helical bundles come together in a kite-like architecture, presenting binding sites for basigin and inhibitory antibodies at one tip. This provides the first structural insight into erythrocyte binding by the Plasmodium RH protein family and identifies novel inhibitory epitopes to guide design of a new generation of vaccines against the blood-stage parasite.

Oncolytic adenovirus expressing bispecific antibody targets T-cell cytotoxicity in cancer biopsies.

Oncolytic viruses exploit the cancer cell phenotype to complete their lytic life cycle, releasing progeny virus to infect nearby cells and repeat the process. We modified the oncolytic group B adenovirus EnAdenotucirev (EnAd) to express a bispecific single‐chain antibody, secreted from infected tumour cells into the microenvironment. This bispecific T‐cell engager (BiTE) binds to EpCAM on target cells and cross‐links them to CD3 on T cells, leading to clustering and activation of both CD4 and CD8 T cells. BiTE transcription can be controlled by the virus major late promoter, limiting expression to cancer cells that are permissive for virus replication. This approach can potentiate the cytotoxicity of EnAd, and we demonstrate using primary pleural effusions and peritoneal malignant ascites that infection of cancer cells with the BiTE‐expressing EnAd leads to activation of endogenous T cells to kill endogenous tumour cells despite the immunosuppressive environment. In this way, we have armed EnAd to combine both direct oncolysis and T cell‐mediated killing, yielding a potent therapeutic that should be readily transferred into the clinic.

Interaction of vanadate with the cloned beta cell K(ATP) channel.

Vanadate is used as a tool to trap magnesium nucleotides in the catalytic site of ATPases. However, it has also been reported to activate ATP-sensitive potassium (KATP) channels in the absence of nucleotides. KATP channels comprise Kir6.2 and sulfonylurea receptor subunits (SUR1 in pancreatic beta cells, SUR2A in cardiac and skeletal muscle, and SUR2B in smooth muscle). We explored the effect of vanadate (2 mm), in the absence and presence of magnesium nucleotides, on different types of cloned KATP channels expressed in Xenopusoocytes. Currents were recorded from inside-out patches. Vanadate inhibited Kir6.2/SUR1 currents by ∼50% but rapidly activated Kir6.2/SUR2A (∼4-fold) and Kir6.2/SUR2B (∼2-fold) currents. Mutations in SUR that abolish channel activation by magnesium nucleotides did not prevent the effects of vanadate. Studies with chimeric SUR indicate that the first six transmembrane domains account for the difference in both the kinetics and the vanadate response of Kir6.2/SUR1 and Kir6.2/SUR2A. Boiling the vanadate solution, which removes the decavanadate polymers, largely abolished both stimulatory and inhibitory actions of vanadate. Our results demonstrate that decavanadate modulates KATP channel activity via the SUR subunit, that this modulation varies with the type of SUR, that it differs from that produced by magnesium nucleotides, and that it involves transmembrane domains 1–6 of SUR.

Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS2, based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of ‘difficult-to-make’ proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen.

Human vaccination against RH5 induces neutralizing antimalarial antibodies that inhibit RH5 invasion complex interactions

The development of a highly effective vaccine remains a key strategic goal to aid the control and eventual eradication of Plasmodium falciparum malaria. In recent years, the reticulocyte-binding protein homolog 5 (RH5) has emerged as the most promising blood-stage P. falciparum candidate antigen to date, capable of conferring protection against stringent challenge in Aotus monkeys. We report on the first clinical trial to our knowledge to assess the RH5 antigen - a dose-escalation phase Ia study in 24 healthy, malaria-naive adult volunteers. We utilized established viral vectors, the replication-deficient chimpanzee adenovirus serotype 63 (ChAd63), and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding RH5 from the 3D7 clone of P. falciparum. Vaccines were administered i.m. in a heterologous prime-boost regimen using an 8-week interval and were well tolerated. Vaccine-induced anti-RH5 serum antibodies exhibited cross-strain functional growth inhibition activity (GIA) in vitro, targeted linear and conformational epitopes within RH5, and inhibited key interactions within the RH5 invasion complex. This is the first time to our knowledge that substantial RH5-specific responses have been induced by immunization in humans, with levels greatly exceeding the serum antibody responses observed in African adults following years of natural malaria exposure. These data support the progression of RH5-based vaccines to human efficacy testing.

Potency of a thermostabilised chimpanzee adenovirus Rift Valley Fever vaccine in cattle.

Development of safe and efficacious vaccines whose potency is unaffected by long-term storage at ambient temperature would obviate major vaccine deployment hurdles and limit wastage associated with breaks in the vaccine cold chain. Here, we evaluated the immunogenicity of a novel chimpanzee adenovirus vectored Rift Valley Fever vaccine (ChAdOx1-GnGc) in cattle, following its thermostabilisation by slow desiccation on glass fiber membranes in the non-reducing sugars trehalose and sucrose. Thermostabilised ChAdOx1-GnGc vaccine stored for 6 months at 25, 37 or 45 °C elicited comparable Rift Valley Fever virus neutralising antibody titres to those elicited by the ‘cold chain’ vaccine (stored at −80 °C throughout) at the same dose, and these were within the range associated with protection against Rift Valley Fever in cattle. The results support the use of sugar-membrane thermostabilised vaccines in target livestock species.

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

Graphical abstract

Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells

Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a leading asexual blood-stage vaccine candidate for malaria. In preparation for clinical trials, a full-length PfRH5 protein vaccine called “RH5.1” was produced as a soluble product under cGMP using the ExpreS2 platform (based on a Drosophila melanogaster S2 stable cell line system). Following development of a high-producing monoclonal S2 cell line, a master cell bank was produced prior to the cGMP campaign. Culture supernatants were processed using C-tag affinity chromatography followed by size exclusion chromatography and virus-reduction filtration. The overall process yielded >400 mg highly pure RH5.1 protein. QC testing showed the MCB and the RH5.1 product met all specified acceptance criteria including those for sterility, purity, and identity. The RH5.1 vaccine product was stored at −80 °C and is stable for over 18 months. Characterization of the protein following formulation in the adjuvant system AS01B showed that RH5.1 is stable in the timeframe needed for clinical vaccine administration, and that there was no discernible impact on the liposomal formulation of AS01B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01B vaccine was immunogenic and could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01B was judged suitable for use in humans and has since progressed to phase I/IIa clinical trial. Our data support the future use of the Drosophila S2 cell and C-tag platform technologies to enable cGMP-compliant biomanufacture of other novel and “difficult-to-express” recombinant protein-based vaccines.Malaria: Successful clinical trial preparation for blood-stage vaccineA vaccine candidate for blood-stage malaria has overcome previous hurdles to enter clinical trials. The protein PfRH5 is an essential blood-stage infection facilitator of malarial parasite Plasmodium falciparum, and a promising target for vaccine strategies. Unfortunately, efforts to produce the protein in an immunogenic, clinically-viable way have been met with difficulty. Here, researchers led by Simon Draper, from the UK’s Jenner Institute, used a fruit fly expression system to produce over 400 mg of high-purity protein. Formulated with an immunity-boosting adjuvant, the vaccine elicited antibodies in mice that proved inhibitory to blood-stage P. falciparum during in vitro assays. The PfRH5 vaccine candidate and its adjuvant have been approved for a clinical trial in the UK, and the authors hope that the expression system used may be beneficial in the expression of other ‘difficult’ proteins.