Improved Virus Neutralization by Plant-produced Anti-HIV Antibodies with a Homogeneous ��1,4-Galactosylated N-Glycan Profile

It is well established that proper N-glycosylation significantly influences the efficacy of monoclonal antibodies (mAbs). However, the specific immunological relevance of individual mAb-associated N-glycan structures is currently largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. Here we report on the generation of a plant-based expression platform allowing the efficient production of mAbs with a homogeneous β1,4-galactosylated N-glycosylation structure, the major N-glycan species present on serum IgG. This was achieved by the expression of a highly active modified version of the human β1,4-galactosyltransferase in glycoengineered plants lacking plant-specific glycosylation. Moreover, we demonstrate that two anti-human immunodeficiency virus mAbs with fully β1,4-galactosylated N-glycans display improved virus neutralization potency when compared with other glycoforms produced in plants and Chinese hamster ovary cells. These findings indicate that mAbs containing such homogeneous N-glycan structures should display improved in vivo activities. Our system, using expression of mAbs in tobacco plants engineered for post-translational protein processing, provides a new means of overcoming the two hurdles that limit the therapeutic use of anti-human immunodeficiency virus mAbs in global health initiatives, low biological potency and high production costs.About 40 million people are estimated to be infected with HIV-1,² and the HIV-1/AIDS epidemic continues to escalate, with the most devastating consequences seen in the most impoverished nations (1). Two strategies that have been pursued over the past 2 decades for stopping the AIDS pandemic/epidemic are the generation of vaccines to prevent HIV infection and the development of microbicides to prevent HIV transmission. Highly effective monoclonal antibodies (mAbs) are suitable to be used in both modalities. To date, only a handful of anti-HIV mAbs with neutralizing activities has been explored in more detail (2). In a recent clinical study, it has been demonstrated that a combination of three broadly neutralizing anti-HIV antibodies (2G12, 2F5, and 4E10) shows promise as AIDS treatment (3). However, despite effective in vitro neutralization activities, relatively modest in vivo effects were obtained, suggesting that the in vivo properties of these antibodies require further improvement (2). Noteworthy, these antibodies bind to HIV envelope proteins thus inhibiting viral entry into target cells (2, 4, 5). In addition to their potential use in therapeutic modalities, this renders them as promising candidates for microbicide development. However, high production costs using mammalian-cell technologies and insufficient efficacy of anti-HIV antibodies are remaining hurdles for their effective use. Among recent advances in generating antibodies with enhanced activities, glyco-engineering has been proven to be a powerful tool (6). It is well established that proper N-glycosylation significantly influences the efficacy of mAbs. Nevertheless, the specific immunological relevance of individual mAb-associated N-glycan structures is largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. A series of studies emphasize the critical role of IgG glycoforms lacking core α1,6-fucose for cell-mediated immunological activities (6). However, the immunological significance of N-glycans with terminal β1,4-galactose residues, the major N-glycan species present on serum IgG, has not yet been established.During the last 2 decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. This resulted in a significant enhancement of expression levels (up to 100-fold) and a reduction of production time (7, 8), which makes the system economically interesting. Another important achievement was the generation of plant glycosylation mutants, which allows a controlled human-type glycosylation of recombinant glycoproteins (9, 10). Recently, we have generated different glycoforms of anti-HIV mAb 2G12 in the tobacco-related plant species Nicotiana benthamiana (9). All of them were functionally active, and HIV neutralization potency was comparable with CHO-derived 2G12. This process involved the generation of a plant glycosylation mutant (ΔXT/FT), which was found to produce mAbs carrying homogeneous N-glycans with terminal N-acetylglucosamine (Gn) residues (i.e. GnGn structures) lacking unwanted plant-specific β1,2-xylose and core α1,3-fucose residues. These glycans are devoid of any β1,4-linked galactose residues; thus in this study, we set out to glyco-engineer ΔXT/FT plants for quantitative β1,4-galactosylation. A highly active modified version of human β1,4-galactosyltransferase was used to transform ΔXT/FT and progeny screened for efficient protein β1,4-galactosylation. In total four glycoforms from the two anti-HIV mAbs 2G12 and 4E10 (plant- and CHO-derived) were generated and compared toward antigen binding and virus neutralization capacities.     

Genetic clonality of Plasmodium falciparum affects the outcome of infection in Anopheles gambiae

Mosquito infections with natural isolates of Plasmodium falciparum are notoriously variable and pose a problem for reliable evaluation of efficiency of transmission-blocking agents for malaria control interventions. Here, we show that monoclonal P. falciparum isolates produce higher parasite loads than mixed ones. Induction of the mosquito immune responses by wounding efficiently decreases Plasmodium numbers in monoclonal infections but fails to do so in infections with two or more parasite genotypes. Our results point to the parasites genetic complexity as a potentially crucial component of mosquito-parasite interactions.     

Miss to the Right: The Effect of Attentional Asymmetries on Goal-Kicking

Cerebral asymmetries for spatial attention generate a bias of attention – causing lines to be bisected to the left or right in near (within reach) and far (outside reach) space, respectively. This study explored whether the rightward deviation for bisecting lines in far space extends to tasks where a ball is aimed between two goal-posts. Kicking was assessed in a laboratory and a real-life setting. In the laboratory setting, 212 participants carried out three conditions: (a) kick a soccer ball at a single goal post, (b) kick a soccer ball between two goal posts and (c) use a stick to indicate the middle between two goal posts. The goals were placed at a distance of 4.0 m. There was no deviation in the one-goal kicking condition – demonstrating that no asymmetries exist in the perceptual motor system when aiming at a single point. When kicking or pointing at the middle between two goal posts, rightward deviations were observed. In the real-world setting, the number of misses to the left or right of goal (behinds) in the Australian Rules football for the 2005–2009 seasons was assessed. The data showed more rightward deviations for kicks at goal. Combined, the studies suggest that the rightward deviation for lines placed in far space extends to the kicking of a football in laboratory and real-life settings. This asymmetry in kicking builds on a body of research showing that attentional asymmetries impact everyday activities.     

A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galβ1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid.     

Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines.     

Characterization of microbial biofilms in a thermophilic biogas system by high-throughput metagenome sequencing

DNAs of two biofilms of a thermophilic two-phase leach-bed biogas reactor fed with rye silage and winter barley straw were sequenced by 454-pyrosequencing technology to assess the biofilm-based microbial community and their genetic potential for anaerobic digestion. The studied biofilms matured on the surface of the substrates in the hydrolysis reactor (HR) and on the packing in the anaerobic filter reactor (AF). The classification of metagenome reads showed Clostridium as most prevalent bacteria in the HR, indicating a predominant role for plant material digestion. Notably, insights into the genetic potential of plant-degrading bacteria were determined as well as further bacterial groups, which may assist Clostridium in carbohydrate degradation. Methanosarcina and Methanothermobacter were determined as most prevalent methanogenic archaea. In consequence, the biofilm-based methanogenesis in this system might be driven by the hydrogenotrophic pathway but also by the aceticlastic methanogenesis depending on metabolite concentrations such as the acetic acid concentration. Moreover, bacteria, which are capable of acetate oxidation in syntrophic interaction with methanogens, were also predicted. Finally, the metagenome analysis unveiled a large number of reads with unidentified microbial origin, indicating that the anaerobic degradation process may also be conducted by up to now unknown species.     

Temperature increases from 55 to 75 °C in a two-phase biogas reactor result in fundamental alterations within the bacterial and archaeal community structure

Agricultural biogas plants were operated in most cases below their optimal performance. An increase in the fermentation temperature and a spatial separation of hydrolysis/acetogenesis and methanogenesis are known strategies in improving and stabilizing biogas production. In this study, the dynamic variability of the bacterial and archaeal community was monitored within a two-phase leach bed biogas reactor supplied with rye silage and straw during a stepwise temperature increase from 55 to 75 °C within the leach bed reactor (LBR), using TRFLP analyses. To identify the terminal restriction fragments that were obtained, bacterial and archaeal 16S rRNA gene libraries were constructed. Above 65 °C, the bacterial community structure changed from being Clostridiales-dominated toward being dominated by members of the Bacteroidales, Clostridiales, and Thermotogales orders. Simultaneously, several changes occurred, including a decrease in the total cell count, degradation rate, and biogas yield along with alterations in the intermediate production. A bioaugmentation with compost at 70 °C led to slight improvements in the reactor performance; these did not persist at 75 °C. However, the archaeal community within the downstream anaerobic filter reactor (AF), operated constantly at 55 °C, altered by the temperature increase in the LBR. At an LBR temperature of 55 °C, members of the Methanobacteriales order were prevalent in the AF, whereas at higher LBR temperatures Methanosarcinales prevailed. Altogether, the best performance of this two-phase reactor was achieved at an LBR temperature of below 65 °C, which indicates that this temperature range has a favorable effect on the microbial community responsible for the production of biogas.