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Global eradication of hepatitis C virus (HCV) infection will require an efficacious vaccine capable of eliciting protective immunity against genetically diverse HCV strains. Natural spontaneous resolution of HCV infection is associated with production of broadly neutralizing antibodies targeting the HCV glycoproteins E1 and E2. As such, production of cross-neutralizing antibodies is an important endpoint for experimental vaccine trials. Varying success generating cross-neutralizing antibodies has been achieved with immunogens derived from naturally-occurring HCV strains. In this study the challenge of minimising the genetic diversity between the vaccine strain and circulating HCV isolates was addressed. Two novel synthetic E2 glycoprotein immunogens (NotC1 and NotC2) were derived from consensus nucleotide sequences deduced from samples of circulating genotype 1 HCV strains. These two synthetic sequences differed in their relative positions in the overall genotype 1a/1b phylogeny. Expression of these constructs in Drosophila melanogaster S2 cells resulted in high yields of correctly-folded, monomeric E2 protein, which were recognised by broadly neutralizing monoclonal antibodies. Immunization of guinea pigs with either of these consensus immunogens, or a comparable protein representing a circulating genotype 1a strain resulted in high titres of cross-reactive anti-E2 antibodies. All immunogens generated antibodies capable of neutralizing the H77 strain, but NotC1 elicited antibodies that more potently neutralized virus entry. These vaccine-induced antibodies neutralized some viruses representing genotype 1, but not strains representing genotype 2 or genotype 3. Thus, while this approach to vaccine design resulted in correctly folded, immunogenic protein, cross-neutralizing epitopes were not preferentially targeted by the host immune response generated by this immunogen. Greater immunofocussing by vaccines to common epitopes is necessary to successfully elicit broadly neutralizing antibodies.

The production of helium bubbles in advanced gas-cooled reactor (AGR) cladding could represent a significant hazard for both the mechanical stability and long-term storage of such materials. However, the high radioactivity of AGR cladding after operation presents a significant barrier to the scientific study of the mechanical properties of helium incorporation, said cladding typically being analyzed in industrial hot cells. An alternative non-active approach is to implant He2+ into unused AGR cladding material via an accelerator. Here, a feasibility study of such a process, using sequential implantations of helium in AGR cladding steel with decreasing energy is carried out to mimic the buildup of He (e.g., 50 appm) that would\ud occur for in-reactor AGR clad in layers of the order of 10 lm in depth, is described. The implanted sample is subsequently analyzed by scanning electron microscopy, nanoindentation, atomic force and ultrasonic force microscopies. As expected, the irradiated zones were affected by implantation damage (

Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230–239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.

More than 170 million people worldwide are chronically infected with hepatitis C virus (HCV) and are at risk of developing liver fibrosis, cirrhosis and hepatocellular carcinoma. Liver transplantation is the only option for patients with HCV-induced end-stage liver diseases. Nevertheless, infection of the newly grafted liver occurs immediately and universally after transplantation. Despite the recent progress in HCV therapy, a prophylactic vaccine is still not available. The role of neutralizing monoclonal antibodies (mAbs) in protection from different viral infections including HCV, HIV and Ebola has been reported. In the last few years, several mAbs with neutralizing activity have been described but only few mAbs have been evaluated in vivo. In the present study, we describe the development of a mAb, designated 2A5, isolated from HCV genotype 1b chronic patient. ELISA results indicated high affinity of mAb 2A5 towards HCV envelope glycoprotein (E1E2). The binding activity was completely lost against denatured E1E2 protein indicating that it targets a conformational epitope within the envelope region. Epitope mapping using alanine mutants of E1E2 proteins defined critical binding residues within the regions 419-447 and 612-617. Results of pseudoparticles (HCVpp) and cell culture produced virus (HCVcc) neutralization showed broad neutralizing activity of mAb 2A5 against all HCV genotypes. The efficacy study of mAb 2A5 in immune-deficient mice of which the liver is repopulated with human hepatocytes (humanized mice) showed complete protection from HCV challenge for genotypes 1a and 4a, while partial protection was achieved for genotypes 1b and 6a. Sequence analysis of E1E2 protein from non-protected mice did not revealed resistance mutations at interaction residues of mAb 2A5. In conclusion, mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence provide an effective strategy to prevent HCV recurrence in chronically infected HCV liver transplant patients. In addition, the broad neutralizing activity of this mAb presents a valuable epitope for the design of HCV vaccine with cross-protection activity.

Immunoadhesins are engineered proteins combining the constant domain (Fc) of an antibody with a ligand-binding (adhesion) domain. They have significant potential as therapeutic agents, because they maintain the favourable pharmacokinetics of antibodies with an expanded repertoire of ligand-binding domains: proteins, peptides, or small molecules. We have recently reported that the addition of a cholesterol group to two HIV antibodies can dramatically improve their antiviral potency. Cholesterol, which can be conjugated at various positions in the antibody, including the constant (Fc) domain, endows the conjugate with affinity for the membrane lipid rafts, thus increasing its concentration at the site where viral entry occurs. Here, we extend this strategy to an HIV immunoadhesin, combining a cholesterol-conjugated Fc domain with the peptide fusion inhibitor C41. The immunoadhesin C41-Fc-chol displayed high affinity for Human Embryonic Kidney (HEK) 293 cells, and when tested on a panel of HIV-1 strains, it was considerably more potent than the unconjugated C41-Fc construct. Potentiation of antiviral activity was comparable to what was previously observed for the cholesterol-conjugated HIV antibodies. Given the key role of cholesterol in lipid raft formation and viral fusion, we expect that the same strategy should be broadly applicable to enveloped viruses, for many of which it is already known the sequence of a peptide fusion inhibitor similar to C41. Moreover, the sequence of heptad repeat-derived fusion inhibitors can often be predicted from genomic information alone, opening a path to immunoadhesins against emerging viruses.

Pamexis hantam Mansell & Ball, sp. nov. Figs 1, 3���5, 9���22, 25, 26, 27 Etymology. Hantam, a noun in apposition, referring to the area in which the new species occurs. The name ��� Hantam ��� is derived from the Khoisan word!Han=ami, also previously written as ���heyntams,��� which means ���where the red bulbs grow��� (TANAP 2016). This refers to a Pelargonium plant with edible roots [probably P. bifolium (Burm.f.) Willd (Germishuizen et al. 2006)]. Description. (Based on holotype male, 12 male, 13 female paratypes) Habitus (Fig. 1). Smallish, robust, broad-winged antlions. Wings pale whitish yellow, with tessellated dark brown markings in forewings and prominent dark brown spots or bands in hind wings, but wing markings in both wings highly variable (Figs 9���10). Head, thorax and abdomen black with yellow markings on thorax. Male ectoprocts short, black, forcipate. Head: black, slightly narrower than prothorax, vertex raised, bi-lobed with long curved black setae dorsally; antennae short, black, strongly clavate, shorter than head width, toruli slightly less than scape-width apart, torular membrane yellow, scape black with long curved setae anteriorly, flagellomeres short, uniformly black, covered with short black setae; eyes characteristically small, less than hemispherical, ocular setae absent but with long curved intermingled black and white setae antero-dorsally, short white setae ventrally and posteriorly; genae shiny black, mandibular articulations yellow, clypeus narrowly black at base, yellow distally, labrum yellowish with two black spots laterally; maxillary and labial palps short, black, terminal labial palpomere spindle-shaped with blunt apex, palpimacula round. Thorax: prothorax almost triangular in dorsal view, with broad black central area and pale spot centrally, lateral margins broadly yellow with two black spots posteriorly, posterior margin black, long black curved setae present along anterior, lateral and posterior margins. Mesoprescutum black with yellow V-shaped mark along posterior margin; mesoscutum black with two large yellow patches anteriorly on either side of midline, two yellow triangular marks at posterior margin and a small yellow spot laterally above each wing base; long black curved setae cover mesonotum, with pale fringe of setae along posterior margin; mesoscutellum black with long white setae along posterior margin. Metaprescutum black, shiny; metascutum black, densely covered with microtrichia, two pale yellow velvety patches present on either side of midline; metascutellum shiny black with row of long white setae along posterior margin. Pleurites and sternites black, covered in long white setae, coxal articular membranes yellow. Wings: short, broad, forewings longer than hind wings, apices rounded, hind margins smooth, membrane pale yellowish-white (greenish in living specimens, fading to pale yellow when preserved); veins pale yellowish-white with variable dark brown markings extending onto membrane (Figs 1, 9, 10) bearing very sparse long white and black setae, hypostigmatic cells long. Forewings: with brown tessellations over most cross-veins, extending onto adjacent membrane; C with short dense black setae, some costal cells biaereolate proximally, pterostigma not discernible, Sc slightly incrassate medially with short setae along distal third, R with row of short curved black setae, slightly longer proximally; Rs arising before Cua fork, 3���7 presectoral crossveins before Rs; Mp2 (oblique vein) arising beyond Cua fork; Cup narrow not fused with A1, A2 and A3 slightly incrassate. Hind wings: pale basally with large spots sometimes coalescing into bands in distal two thirds; origin of Rs proximal to fork of Mp fork; 1 presectoral crossvein, Mp incrassate before fork; incrassate Cua arches forward at junction with posterior branch of Mp, forming the typical palparine recurrent vein; 1A incrassate. Legs: long, black; hind legs extending to abdominal segment 4; tibiae longer than tarsi in all legs, tibial spurs robust, curved, extending to middle of T2; T1 ��� T4 short, T5 long, approximately equal to combined length of T1��� T4; pretarsal claws longer than T1, black, slightly curved. Abdomen: stout, slightly shorter than hind wings, completely black in male (Fig. 25), covered with short black setae, in female usually black with yellow distal margins (Fig. 26). Males with tergite 9 divided (Figs 11���12), sternite IX (Fig. 13) with acute but smooth apex. Ectoprocts (Figs 11���13) curved, cylindrical, with slender spines distally, long slender setae proximally, two stout spines basally; gonarcus and parameres (Figs 14���16) fused into a rigid cone-shaped structure, parameres slerotized, shiny black with medial tuft of sensory setae (Fig. 14), gonarcal bulla (Figs 14���16) translucent; hypandrium internum (Figs 17���19) lightly sclerotized, keel-shaped. Females (Fig. 20) with rounded ectoprocts bearing stout fossorial spines; lateral gonapohyses rounded with stout fossorial spines; anterior gonapophyses rounded with long slender setae; pregenitalae (Fig. 22) triangular, sclerotized; spermathecae (Fig. 21) slender, tapering distally. The small sclerotized triangular pregenitale of the female, which is situated in membranous folds between the anterior gonapophyses serves to accommodate and guide the equally inconspicuous keel-shaped hypandrium internum of the male to the spermatheca during sperm transfer. Larvae: unknown. Distribution. Northern Cape Province, South Africa. Systematic position. Pamexis hantam is morphologically similar to P. namaqua (Fig. 2) and they are clearly adelphotaxa. Mansell (1992) stated that P. namaqua was most closely related to P. contaminatus Hagen, but this relationship has now been superseded by discovery of P. hantam, which is clearly closer to P. namaqua than to P. contaminatus. Pamexis hantam is distinguished from P. namaqua by its larger size and wing length and by the markings on the thorax and black abdomen (Figs 25, 26). The wings of P. hantam are longer and less rounded than those of P. namaqua, and also lack the diffuse brown suffusion manifest in P. namaqua (Fig. 2). The males are easily separated by the black ectoprocts of P. hantam compared to the yellow ectoprocts of P. namaqua (Fig. 23) and the extensive yellow markings on the abdomen of the latter (Figs 23, 24). There appears to be no overlap in the currently known distributions of the two taxa and the adults also have disparate phenologies. Habitat and behaviour. This species occurs over a recorded range of about 25 km from the summit of the Hantam Mountain (Hantamberg) (over 1500 m) southwards to Keiskie Pass and beyond (at 1220 m). The vegetation type on the Hantam Mountain is Hantam Plateau Dolerite Renosterveld (FRd2), while the vegetation type near Keiskie Pass is Roggeveld Shale Renosterveld (FRs3) (Mucina & Rutherford 2006). Both are in the Fynbos Biome. This Pamexis species occurs at the highest altitude (1200���1500 m) of the genus, where the frost incidence is 10���40 days per year. Summers are very hot, with a mean daily maximum temperature of 31.1�� for February (Mucina & Rutherford 2006). The unifying feature of all the known localities is the presence of white and black crustose lichens on the rocky landscape with low shrubland and rich herblands containing an abundance of geophytes (Mucina & Rutherford 2006). When settled on the crustose lichens on rocks, these alert insects are exceptionally difficult to detect. The thallus of the lichens is a light whitish/green colour, with the discs of the cup-shaped, lecideine apothecia, being black. These colours are consequently very similar to those of P. hantam, with the rounded dark coloration of the fruiting bodies (apothecia) also being very similar in size and shape to many of the ovoid black markings on the fore- and hind-wings (Figs 3���5). The crypsis/camouflage is enhanced by the extensive pale setae on the head and thorax, which minimize an edge shadow from the visible portion of the body when resting on the lichen covered rocks during the day (see Fig. 4). When disturbed, these rapid-flying diurnal antlions take to the air and are carried by the prevailing winds, settling up to 70 meters away. They usually settle on rocks or the ground. They occasionally also settle on vegetation. Many of the low shrubs in the area of occurrence have grey/green leaves, including Eriocephalus sp. (���kapokbossie���) and the ���renosterbos��� (Elytropappus rhinocerotis), again enhancing camouflage. In flight they strongly resemble the local and very widespread butterfly Belenois aurota aurota, the brown-veined white (family Pieridae). The long flight episodes possibly lessen the chance of asilid attack. The rapid transition from a settled and camouflaged insect on a rock to a white flash-pattern on flight is probably an effective anti-predation ���startling��� attribute. No oviposition sites nor early stages of any of the taxa in this genus are currently known. Material examined. Holotype ♂, NEUR 10999, SOUTH AFRICA, Northern Cape Province, Keiskieberg Pass (31.35.26S 19.51.52E, 1176m), 18.xii.2010, M.W. Mansell & J.B. Ball (SANC). Paratypes: 1♀, same data as holotype (SANC); 2♂ 1♀, NEUR 10706, same locality but 16.xii.2009, J.B. Ball (JBBC & SANC); 1♂ 1♀, NEUR09689, Hantamberg (31.20.03S 19.48.07E, 1533m), 16.xii.2004, J. White (JBBC); 1♂ 2♀, JBNE 00541, same data but 20.xii.2006 (JBBC); 4♂ 5♀, NEUR 09761, same data but 21.xii.2006 (JBBC); 1♂ 2♀, NEUR 11001, same locality but 19.xii.2010, M.W. Mansell & J.B. Ball (SANC); 1♂, NEUR11000, Keiskie Farm, 31.39.12S 19.53.51E, 1254m, 18.xii.2010, M.W. Mansell & J.B. Ball (SANC); 2♂ 1♀, NEUR 11075, same data but 10.i.2011, A.P. Marais (SANC). Additional material: 1♀ (Photograph), Akkerendam Nature Reserve, Calvinia (31.25.38S 19.46.57E), 18.xii.2014, C.K. Willis (VM279)*; 1♀ (Photograph), same data and photographer but 23.xii.2015 (VM9363). * VM refers to the Virtual Museum Number, Animal Demography Unit, University of Cape Town: http:// vmus.adu.org.za/?vm=LacewingMAP-279 and 9363., Published as part of Mansell, Mervyn W. & Ball, Jonathan B., 2016, A remarkable new lichenophilous Pamexis species from the Hantam Karoo of South Africa (Neuroptera: Myrmeleontidae: Palparini), pp. 171-183 in Zootaxa 4184 (1) on pages 172-182, DOI: 10.11646/zootaxa.4184.1.11, http://zenodo.org/record/164566, {"references":["Germishuizen, G., Meyer, N. L., Steenkamp, Y. & Keith, M. (Eds.) (2006) A checklist of South African plants. Southern African Botanical Diversity Network Report No. 41. SABONET, Pretoria. pp. 1 - 1126.","Mansell, M. W. (1992) The ant-lions of southern Africa: genus Pamexis Hagen (Neuroptera: Myrmeleontidae: Palparinae: Palparini). Systematic Entomology, 17, 65 - 78. http: // dx. doi. org / 10.1111 / j. 1365 - 3113.1992. tb 00322. x","Mucina, L. & Rutherford, M. C. (Eds.) (2006) The vegetation of South Africa, Lesotho and Swaziland. Strelitzia 19. South African National Biodiversity Institute, Pretoria, pp. 1 - 807."]}

The 2013–2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the cocirculation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation - driving cytokine release and Granzyme B upregulation - is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

A novel and broadly applicable strategy combining site-directed mutagenesis and DNA assembly for constructing seamless viral chimeras is described using hepatitis C virus (HCV) as an exemplar. Full-length HCV genomic cloning cassettes, which contained flexibly situated restriction endonuclease sites, were prepared via a single, site-directed mutagenesis reaction and digested to receive PCR-amplified virus envelope genes by In-Fusion cloning. Using this method, we were able to construct gene-shuttle cassettes for generation of cell culture-infectious JFH-1-based chimeras containing genotype 1-3 E1E2 genes. Importantly, using this method we also show that E1E2 clones that were not able to support cell entry in the HCV pseudoparticle assay did confer entry when shuttled into the chimeric cell culture chimera system. This method can be easily applied to other genes of study and other viruses and, as such, will greatly simplify reverse genetics studies of variable viruses.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver carcinoma and new therapies based on novel targets are needed. The tight junction protein claudin 1 (CLDN-1) is essential for HCV cell entry and spread, and anti-CLDN-1 rat and mouse mAbs are safe and effective in preventing and treating HCV infection in a human liver chimeric mouse model. To accelerate translation of these observations into a novel approach to treat HCV infection and disease in humans, we screened a phage display library of human single-chain antibody fragments by using a panel of CLDN-1-positive and -negative cell lines and identified phage specifically binding to CLDN-1. The 12 clones showing the highest levels of binding were converted into human IgG4. Some of these mAbs displayed low-nanomolar affinity, and inhibited infection of human hepatoma Huh7.5 cells by different HCV isolates in a dose-dependent manner. Cross-competition experiments identified six inhibitory mAbs that recognized distinct epitopes. Combination of the human anti-SRB1 mAb C-1671 with these anti-CLDN-1 mAbs could either increase or reduce inhibition of cell culture-derived HCV infection in vitro. These novel human anti-CLDN-1 mAbs are potentially useful to develop a new strategy for anti-HCV therapy and lend support to the combined use of antibodies targeting the HCV receptors CLDN-1 and SRB1, but indicate that care must be taken in selecting the proper combination.