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The COVID-19 pandemic has been recognized as the most significant global outbreak in recent decades. While several vaccines have been developed and administered worldwide, they face persistent challenges such as declining immunity and emergence of new viral strains that can evade them. To combat these obstacles, it is crucial to develop new antiviral treatments and vaccines that target highly conserved regions of the virus. One promising therapeutic approach against SARS- CoV-2 is to disrupt the membrane fusion mechanism that enables the virus to enter the cell. This process is promoted by the Spike (S) protein, which is a trimer of heterodimers (with S1 and S2 subunits), and involves the interaction between two highly conserved heptad-repeat regions (HR1 and HR2) of S2 to form a 6-helix bundle (6HB) structure. Our previous research with HIV-1, a virus that shares a similar fusion mechanism with SARS-CoV-2, has demonstrated that single-chain chimeric proteins that mimic the HR1 region of the gp41 subunit, functionally equivalent to SARS-CoV-2 S2 subunit, can bind strongly HR2 and constitute potent fusion inhibitors [1]. In this work, we describe the design, engineering, and biophysical characterization of a series of single-chain proteins (named CoVS-HR1) that imitate the HR1 region in its trimeric coiled-coil conformation. While a first generation of proteins folded in-helical structures as expected, they tended to oligomerize and some of them showed low stability [2], a second generation of re-engineered proteins showed much better biophysical characteristics in terms of structure, stability and oligomerization state and allowed a detailed study of their binding to their complementary HR2 peptides [3]. Finally, we present the high-resolution crystallographic structure of the complex between a CoVS-HR1 protein and an HR2 peptide, which allowed a deep analysis of the interaction and highlighted the most implicated residues. It also allowed us to identify several int

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.

Olea europaea subsp. cuspidata has a relatively low commercial value due to the low size and pulp to stone ratio of its drupes compared to commercial olive cultivars. Nevertheless, this subspecies could represent a valid source of useful traits for olive breeding. In the current work, the drupe metabolic composition (secoiridoids, flavonoids, simple phenols, triterpenic acids, etc.) of a progeny of 27 cuspidata genotypes coming from free pollination and their female parent was evaluated by applying a powerful LC-MS method. A total of 62 compounds were detected within the profiles; 60 of them were annotated and 27 quantified. From a quantitative point of view, the genotypes from the progeny of cuspidata showed quite different metabolic profiles to olive common cultivars ("Arbequina", "Frantoio", "Koroneiki" and "Picual") used as controls. Cuspidata drupes were richer in terms of several bioactive compounds such as rutin, hydroxytyrosol glucoside, a few interesting secoiridoids and the compounds of m/z 421 and 363. The relationships among several secondary metabolites determined in the progeny inferred from the results of both PCA and cross-correlation analysis were explained according to metabolic biosynthesis pathways in olive drupes. These outcomes underlined the potential of cuspidata genetic resources as a source of potentially interesting variability in olive breeding programs.