Hamster neogenin, a host-cell protein contained in a respiratory syncytial virus candidate vaccine, induces antibody responses in rabbits but not in clinical trial participants.

A recombinant respiratory syncytial virus (RSV) fusion glycoprotein candidate vaccine (RSV-PreF) manufactured in Chinese hamster ovary cells was developed for immunization of pregnant women, to protect newborns against RSV disease through trans-placental antibody transfer. Traces of a host-cell protein, hamster neogenin (haNEO1), were identified in purified RSV-PreF antigen material. Given the high amino-acid sequence homology between haNEO1 and human neogenin (huNEO1), there was a risk that potential vaccine-induced anti-neogenin immunity could affect huNEO1 function in mother or fetus. Anti-huNEO1 IgGs were measured by enzyme-linked immunosorbent assay in sera from rabbits and trial participants (Phase 1 and 2 trials enrolling 128 men and 500 non-pregnant women, respectively; NCT01905215/NCT02360475) collected after immunization with RSV-PreF formulations containing different antigen doses with/without aluminum-hydroxide adjuvant. In rabbits, four injections administered at 14-day intervals induced huNEO1-specific IgG responses in an antigen-dose- and adjuvant-dependent manner, which plateaued in the highest-dose groups after three injections. In humans, no vaccination-induced anti-huNEO1 IgG responses were detected upon a single immunization, as the values in vaccine and control groups fluctuated around pre-vaccination levels up to 90/360 days post-vaccination. A minority of participants had anti-huNEO1 levels ≥ assay cutoff before vaccination, which did not increase post-vaccination. Thus, despite detecting vaccine-induced huNEO1-specific responses in rabbits, we found no evidence that the candidate vaccine had induced anti-huNEO1 immunity in human adults. The antigen purification process was nevertheless optimized, and haNEO1-reduced vaccines were used in a subsequent Phase 2 trial enrolling 400 non-pregnant women (NCT02956837), in which again no vaccine-induced anti-huNEO1 responses were detected.

Localization of the O-glycosylated sites in peptides by fixed-charge derivatization with a phosphonium group.

The present study demonstrates that matrix-assisted laser desorption ionization/postsource decay (MALDI/PSD) analysis of the molecular cation of glycopeptides derivatized at their amino terminus with a phosphonium group cleaves peptide backbone without removing the glycan. The predictable a-type fragment ions retain the glycan moiety, enabling unambiguous localization of O-glycans on the peptide chain. In contrast, collision-activated dissociation tandem mass spectrometry analysis carried out on the doubly charged protonated phosphonium cation results in the predominant loss of the sugar moiety from the peptide. This result supports the previously proposed charge-induced fragmentation mechanism of the sugar-peptide bond. MALDI/PSD analysis of glycopeptides converted to their acetyl phosphonium derivatives is an effective alternative to electron capture dissociation, as illustrated by the positioning of up to three GalNac residues along the full tandem repeat peptide sequence derived from the MUC 5AC mucin.

Molecular fingerprinting of carbohydrate structure phenotypes of three porifera proteoglycan-like glyconectins.

Glyconectins (GNs) represent a new class of proteoglycan-like cell adhesion and recognition molecules found in several Porifera species. Physico-chemical properties of GN carbohydrate moieties, such as size, composition, and resistance to most glycosaminoglycan-degrading enzymes, distinguish them from any other type of known glycoproteins. The molecular mechanism of GN-mediated self/non-self discrimination function is based on highly species-specific and Ca(2+)-dependent GN to GN associations that approach the selectivity of the evolutionarily advanced immunoglobulin superfamily. Carbohydrates of glyconectins 1, 2, and 3 are essential for species-specific auto-aggregation properties in three respective Porifera species. To obtain a structural insight into the molecular mechanisms, we performed carbohydrate structural analyses of glyconectins isolated from the three sponge model systems, Microciona prolifera (GN1), Halichondria panicea (GN2), and Cliona celata (GN3). The glycan content of all three GNs ranged between 40 and 60% of their total mass. Our approach using sequential and selective chemical degradation of GN glycans and subsequent mass spectrometric and NMR analyses revealed that each glyconectin presents novel and highly species-specific carbohydrate sequences. All three GNs include distinct acid-resistant and acid-labile carbohydrate domains, the latter composed of novel repetitive units. We have sequenced four short sulfated and one pyruvilated unit in GN1, eight larger and branched pyruvilated oligosaccharides in GN2, which represent a heterogeneous but related family of structures, and four sulfated units in GN3.

Thioredoxin post-transcriptional regulation by H19 provides a new function to mRNA-like non-coding RNA.

Classically, the functional product of coding genes is a protein whose synthesis is directed by an mRNA-template. However, in the last few years several genes yielding an mRNA-like non-coding RNA as a functional product have been identified. In most cases these transcripts are synthesized by the RNA polymerase II, capped, spliced and polyadenylated, like classical mRNA. These latter have non-conserved open reading frames and seem to be untranslated. Consequently, it has been proposed and admitted that these genes act at the RNA level, and are so-called 'riboregulators'. H19 belongs to this class of gene and its role remains a matter of debate: for some authors it is an oncogene, for others a tumour suppressor. Here, we demonstrate, using a proteomic approach, that an H19 overexpression in human cancerous mammary epithelial cells stably transfected with genomic DNA containing the entire H19 gene is responsible for positively regulating at the post-transcriptional level the thioredoxin, a key protein of the cellular redox metabolism. Interestingly, this protein accumulates in many cancerous tissues, such as breast carcinomas in which we have also demonstrated an overexpression of the H19 gene.

Caffeoyl-coenzyme A 3-O-methyltransferase enzyme activity, protein and transcript accumulation in flax (Linum usitatissimum) stem during development.

Flax (Linum usitatissimum) is a commercially important fiber crop in Europe. Lignification of its phloem fibers, although weak, causes a decrease in their commercial quality. In flax, fiber lignin mainly consists of guaiacyl (G) units in contrast to the mixed guaiacyl-syringyl (G-S) lignin type occurring in xylem fibers. G lignins are reported as more condensed polymers due to a higher frequency of 5-5 linkages, whereas the deposition of syringyl end groups in lignins increases the proportion of alky-aryl ether linkages as beta-O-4-bonds. The type of linkages within a lignin polymer depends on the methylation of either the 3-hydroxyl groups or both 3-OH and 5-OH groups, which is controlled by two enzymes: caffeate 3-O-methyltransferase (COMT) and caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT). First, we measured the in vitro activity of both OMTs in the flax stem tissues during stem development. CCoAOMT activity varied in the same way as COMT, i.e. increased gradually with stem maturity, from the top to the bottom of the stem, was maximum at the flowering stage and was lower, but still scorable, in the outer fiber-bearing tissues than in the xylem cells. In a second step, we focused our studies on the characterization of CCoAOMT in order to understand the implication of this enzyme in the lignification of flax fiber cells. CCoAOMT activity appeared to be associated with the accumulation of an acidic 33-kDa polypeptide identified as a CCoAOMT after immunological cross-reactivity with a poplar CCoAOMT and microsequencing. The differential accumulation of the CCoAOMT protein was confirmed by immunolocalization on tissue prints and correlated with that of the transcripts, suggesting a transcriptional regulation of CCoAOMT in the flax stem.