In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants.

N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics, including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e., effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

Plant-made E2 glycoprotein single-dose vaccine protects pigs against classical swine fever.

Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programmes and culling of infected or at-risk animals, often at very high cost. Current CSFV-modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programmes. Alternatively, first-generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide-spread deployment. To address the issues of scalability, cost of production and immunogenicity, we have employed an Agrobacterium-mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil-in-water emulsion adjuvants. We report the manufacturing of adjuvanted, plant-made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single-dose vaccination, which was accompanied by strong virus neutralization antibody responses.

The evolutionary genetics of the genes underlying phenotypic associations for loblolly pine (Pinus taeda, Pinaceae).

A primary goal of evolutionary genetics is to discover and explain the genetic basis of fitness-related traits and how this genetic basis evolves within natural populations. Unprecedented technological advances have fueled the discovery of genetic variants associated with ecologically relevant phenotypes in many different life forms, as well as the ability to scan genomes for deviations from selectively neutral models of evolution. Theoretically, the degree of overlap between lists of genomic regions identified using each approach is related to the genetic architecture of fitness-related traits and the strength and type of natural selection molding variation at these traits within natural populations. Here we address for the first time in a plant the degree of overlap between these lists, using patterns of nucleotide diversity and divergence for >7000 unique amplicons described from the extensive expressed sequence tag libraries generated for loblolly pine (Pinus taeda L.) in combination with the >1000 published genetic associations. We show that loci associated with phenotypic traits are distinct with regard to neutral expectations. Phenotypes measured at the whole plant level (e.g., disease resistance) exhibit an approximately twofold increase in the proportion of adaptive nonsynonymous substitutions over the genome-wide average. As expected for polygenic traits, these signals were apparent only when loci were considered at the level of functional sets. The ramifications of this result are discussed in light of the continued efforts to dissect the genetic basis of quantitative traits.

Association of loblolly pine xylem development gene expression with single-nucleotide polymorphisms.

Variation in the expression of genes with putative roles in wood development was associated with single-nucleotide polymorphisms (SNPs) using a population of loblolly pine (Pinus taeda L.) that included individuals from much of the native range. Association studies were performed using 3938 SNPs and expression data obtained using quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) for 106 xylem development genes in 400 clonally replicated loblolly pine individuals. A general linear model (GLM) approach, which takes the underlying population structure into consideration, was used to discover significant associations. After adjustment for multiple testing using a false discovery rate correction, 88 statistically significant associations (Q<0.05) were observed for 80 SNPs with the expression data of 33 xylem development genes. Thirty SNPs caused nonsynonymous mutations, 18 resulted in synonymous mutations, 11 were in 3' untranslated regions (UTRs), 1 was in a 5' UTR and 20 were in introns. Using AraNet, we found that Arabidopsis genes with high similarity to the loblolly pine genes involved in 21 of the 88 statistically significant associations are connected in functional gene networks. Comparisons of gene expression values revealed that in most cases the average expression in plants homozygous for the rare SNP allele was lower than that of plants that were heterozygous or homozygous for the abundant allele. Although there are association studies of SNPs and expression profiles for humans, Arabidopsis and white spruce, to the best of our knowledge, this is the first example of such an association genetic study in pines. Functional validation of these associations will lead to a deeper understanding of the molecular basis of phenotypic differences in wood development among individuals in conifer populations.

RNAi-mediated Ultra-low gossypol cottonseed trait: performance of transgenic lines under field conditions.

Cottonseed remains a low-value by-product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra-low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ-cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild-type levels of gossypol and related terpenoids. Although it was a relatively small-scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild-type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%-8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi-based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever-increasing needs of humanity.

Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination.

Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.