Zika virus-like particle vaccine fusion loop mutation increases production yield but fails to protect AG129 mice against Zika virus challenge

Zika virus (ZIKV) is a mosquito-borne flavivirus with maternal infection associated with preterm birth, congenital malformations, and fetal death, and adult infection associated with Guillain-Barré syndrome. Recent widespread endemic transmission of ZIKV and the potential for future outbreaks necessitate the development of an effective vaccine. We developed a ZIKV vaccine candidate based on virus-like-particles (VLPs) generated following transfection of mammalian HEK293T cells using a plasmid encoding the pre-membrane/membrane (prM/M) and envelope (E) structural protein genes. VLPs were collected from cell culture supernatant and purified by column chromatography with yields of approximately 1-2mg/L. To promote increased particle yields, a single amino acid change of phenylalanine to alanine was made in the E fusion loop at position 108 (F108A) of the lead VLP vaccine candidate. This mutation resulted in a modest 2-fold increase in F108A VLP production with no detectable prM processing by furin to a mature particle, in contrast to the lead candidate (parent). To evaluate immunogenicity and efficacy, AG129 mice were immunized with a dose titration of either the immature F108A or lead VLP (each alum adjuvanted). The resulting VLP-specific binding antibody (Ab) levels were comparable. However, geometric mean neutralizing Ab (nAb) titers using a recombinant ZIKV reporter were significantly lower with F108A immunization compared to lead. After virus challenge, all lead VLP-immunized groups showed a significant 3- to 4-Log₁₀ reduction in mean ZIKV RNAemia levels compared with control mice immunized only with alum, but the RNAemia reduction of 0.5 Log₁₀ for F108A groups was statistically similar to the control. Successful viral control by the lead VLP candidate following challenge supports further vaccine development for this candidate. Notably, nAb titer levels in the lead, but not F108A, VLP-immunized mice inversely correlated with RNAemia. Further evaluation of sera by an in vitro Ab-dependent enhancement assay demonstrated that the F108A VLP-induced immune sera had a significantly higher capacity to promote ZIKV infection in FcγR-expressing cells. These data indicate that a single amino acid change in the fusion loop resulted in increased VLP yields but that the immature F108A particles were significantly diminished in their capacity to induce nAbs and provide protection against ZIKV challenge.     

Population history and genetic structure of a circumpolar species: the arctic fox

The circumpolar arctic fox Alopex lagopus thrives in cold climates and has a high migration rate involving long-distance movements. Thus, it differs from many temperate taxa that were subjected to cyclical restriction in glacial refugia during the Ice Ages. We investigated population history and genetic structure through mitochondrial control region variation in 191 arctic foxes from throughout the arctic. Several haplotypes had a Holarctic distribution and no phylogeographical structure was found. Furthermore, there was no difference in haplotype diversity between populations inhabiting previously glaciated and unglaciated regions. This suggests current gene flow among the studied populations, with the exception of those in Iceland, which is surrounded by year-round open water. Arctic foxes have often been separated into two ecotypes: 'lemming' and 'coastal'. An analysis of molecular variance suggested particularly high gene flow among populations of the 'lemming' ecotype. This could be explained by their higher migration rate and reduced fitness in migrants between ecotypes. A mismatch analysis indicated a sudden expansion in population size around 118 000 BP, which coincides with the last interglacial. We propose that glacial cycles affected the arctic fox in a way opposite to their effect on temperate species, with interglacials leading to short-term isolation in northern refugia.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 79–89.     

Identification of vascular endothelial cells in murine omentum using the lectin, Dolichos biflorus agglutinin: possible applications in the study of angiogenesis

Summary The purpose of this study was to determine whether the plant lectin,Dolichos biflorus agglutinin (DBA), can be used to recognize capillary endothelial cells and their processes during angiogenesis. By means of a peroxidase conjugate of DBA, blood vessels were visualized in whole mounts and ultrathin sections of mouse omentum. A part of this mesentery normally comprises an avascular membrane that is approximately 30 µm in thickness. Changes in the vascular plexus bordering this membrane were induced by intraperitoneal injection of irradiated Landschutz cells. Vascular endothelial cells were precisely and intensely stained, and vasculogenic processes were reliably distinguished from those of other cells. This technique permitted observation of the structure and distribution of capillary sprouts, and their relationship to each other and to pre-existing blood vessels. It was discovered that filiform projections extend from sprout apices. These projections may fuse allowing adjacent sprouts to form a new capillary loop.     

Pre-Clinical Development of a Recombinant,Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

Background

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.

Methods

The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.

Results

Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.

Conclusions

The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.     

Notes On The Occurrence Of Clathrus Cancellatus,Tournf., In Argyllshire

Background: Estuaries are characterised by salinity gradients and regular flooding events. These environmental factors form stress gradients, along which species composition changes.

Aims: Analyse and compare patterns of plant species diversity along the estuarine salinity and flooding gradients of the Elbe and Connecticut Rivers.

Methods: Vegetation was sampled at three elevations (low, mid, high) in five sites of each marsh type (fresh, brackish, salt) in both estuaries. Patterns of species density (SD) and evenness (E) along the gradients were analysed and compared between the two estuaries with three-factor ANOVAs.

Results: The regional species pool was 33% higher for the Connecticut than for the Elbe. SD of fresh marshes (19 ± 2.2) was more than twice in the Connecticut than in the Elbe. We found an overall increase in SD from low to high elevation and from salt to freshwater marshes in both estuaries. However, SD and E were strongly depressed at intermediate elevations in the Elbe fresh and brackish marshes.

Conclusions: Although diversity patterns in the two estuaries show overall similarities, patterns of SD and E differ, when particular elevational zones and marsh types are compared. We hypothesise this to be due to evolutionary and historical influences on the regional species pools, shaping the impact of local biotic and abiotic processes.