Suicide attempts in French Polynesia during the era of COVID-19: a prospective analysis over three years.

Background: Past studies in French Polynesia have identified suicide as a significant concern, with a measured annual incidence of 79.4 attempts per 100,000 population during 2008-2010. In response to the COVID-19 pandemic, a monitoring system was established to track and investigate suicide attempts (SA). Methods: A prospective study was conducted between April 2020 and March 2023, including all patients referred to the French Polynesia Hospital Center for SA. Demographic factors as well as clinical parameters were analyzed. Findings: During the study period, 895 SAs were registered and confirmed, with a crude annual rate of 106.7 events and the adjusted rate at 113.2 per 100,000 population. Substantial majority of SA happened in the island of Tahiti. Half of the subjects did not have psychiatric diagnosis. There was a significant increase in SA from year 1 to year 3, with young people (female more than male) particularly at risk, especially in Tahiti. The normalized incidence among females younger than 20-year-old was as high as 310.4 per 100,000 population. Interpretation: Our data revealed an overall 34.4% increase in SA in French Polynesia, with a striking 54.9% increase during the third year of pandemic. The last year's record high incidence, is confirmed by increased activity on suicide hotlines, notably in Tahiti. A correlation between COVID exposure and suicidal behaviors, both at the individual and social level, is suspected with young female in Tahiti being the most vulnerable. These findings highlight the need for reinforced prevention and an efficient suicide monitoring system even after the public health emergency was declared over. Funding: The study is investigator-initiated without funding.

The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).

Atypical antigen recognition mode of a shark immunoglobulin new antigen receptor (IgNAR) variable domain characterized by humanization and structural analysis.

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.

Engineering a human-like glycosylation to produce therapeutic glycoproteins based on 6-linked sialylation in CHO cells.

When recombinant glycoproteins for therapeutic use are to be produced on an industrial scale, there is a crucial need for technologies that can engineer fast-growing stable cells secreting the protein drug at a high rate and with a defined and safe glycosylation profile. Current cell lines approved for drug production are essentially from rodent origin. Their glycosylation machinery often adds undesired carbohydrate determinants which may alter protein folding, induce immunogenicity, and reduce circulatory life span of the drug. Notably, sialic acid as N-acetylneuraminic acid is not efficiently added in most mammalian cells and the 6-linkage is missing in rodent cells. Engineering cells with the various enzymatic activities required for sialic acid transfer has not yet succeeded in providing a human-like pattern of glycoforms to protein drugs. To date, there is a need for engineering animal cells and get highly sialylated products that resemble as closely as possible to human proteins. We have designed ST6Gal minigenes to optimize the ST6GalI sialyltransferase activity and used them to engineer ST6(+)CHO cells. When stably transfected in cells expressing a protein of interest or not, these constructs have proven to equip cell clones with efficient transfer activity of 6-linked sialic acid. In this chapter, we describe a methodology for generating healthy stable adherent clones with hypersialylation activity and high secretion rate.

Oligomerization is required for the activity of recombinant soluble LOX-1.

LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.

FN3: a new protein scaffold reaches the clinic.

In the ten years since the first fibronectin type III (FN3) domain library was published, FN3 has continued to show promise as a scaffold for the generation of stable protein domains that bind to targets with high affinity. A variety of display systems, library designs and affinity maturation strategies have been used to generate FN3 domains with nanomolar to picomolar affinities. The first crystal structures of engineered FN3 molecules in complex with their targets have been solved, and structural studies of engineered FN3 have begun to reveal determinants of stability and to define zones that accept mutations with minimal trade-off between affinity and stability. CT-322, the first engineered FN3 to enter clinical development, is now entering Phase II trials for glioblastoma multiforme.

A single intermolecular contact mediates intramolecular stabilization of both RNA and protein.

An arginine-rich peptide from the Jembrana disease virus (JDV) Tat protein is a structural "chameleon" that binds bovine immunodeficiency virus (BIV) or HIV TAR RNAs in two different binding modes, with an affinity for BIV TAR even higher than the cognate BIV peptide. We determined the NMR structure of the JDV Tat-BIV TAR high-affinity complex and found that the C-terminal tyrosine in JDV Tat forms a network of inter- and intramolecular hydrogen bonding and stacking interactions that simultaneously stabilize the beta-hairpin conformation of the peptide and a base triple in the RNA. A neighboring histidine also appears to help stabilize the peptide conformation. Induced fit binding is recurrent in protein-protein and protein-nucleic acid interactions, and the JDV Tat complex demonstrates how high affinity can be achieved not only by optimization of the binding interface but also by inducing new intramolecular contacts that stabilize each binding partner. Comparison to the cognate BIV Tat peptide-TAR complex shows how such a costabilization mechanism can evolve with only small changes to the peptide sequence. In addition, the bound structure of BIV TAR in the chameleon peptide complex is strikingly similar to the bound conformation of HIV TAR, suggesting new strategies for the development of HIV TAR binding molecules.

Viral RNA gymnastics.

Retroviruses have a stretch of RNA that dimerizes during viral particle formation. A new study suggests that RNA flexibility in the monomeric form may facilitate dimerization or other RNA-dependent viral functions.

Selection of TAR RNA-binding chameleon peptides by using a retroviral replication system.

The interaction between the arginine-rich motif (ARM) of the human immunodeficiency virus (HIV) Tat protein and TAR RNA is essential for Tat activation and viral replication. Two related lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), also require Tat ARM-TAR interactions to mediate activation, but the viruses have evolved different RNA-binding strategies. Interestingly, the JDV ARM can act as a "chameleon," adopting both the HIV and BIV TAR binding modes. To examine how RNA-protein interactions may evolve in a viral context and possibly to identify peptides that recognize HIV TAR in novel ways, we devised a retroviral system based on HIV replication to amplify and select for RNA binders. We constructed a combinatorial peptide library based on the BIV Tat ARM and identified peptides that, like the JDV Tat ARM, also function through HIV TAR, revealing unexpected sequence characteristics of an RNA-binding chameleon. The results suggest that a retroviral screening approach may help identify high-affinity TAR binders and may provide new insights into the evolution of RNA-protein interactions.