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Effective vaccines in preventing HIV-1 infection should induce broadly neutralizing antibodies (bNAbs), active against most of the HIV-1 quasispecies circulating in infected individuals worldwide. Such bNAbs developed in 1-2% of infected individuals. Their passive administration has been shown to be effective in preclinical and clinical settings. In this work, we aimed at understanding the mechanisms by which IgG and IgA bNAbs can interfere with the transmission and propagation of HIV-1. Two research avenues have been investigated: First, the study of the role of anti-HIV-1 antibodies in the protection of two tissue barriers of importance in HIV-1 infection: the epithelium of the female genital tract and the endothelium of the blood-brain barrier. Using viral transmigration systems based on culture monolayers coupled with confocal microscopy analyses, we show that the HIV-1 envelope (Env) is not (in the epithelium) or partially required (in the endothelium) for an optimal viral transcytosis. Moreover, we find that none of the bNAbs and non- or weakly neutralizing antibodies tested can block the passage of the virus through the two barriers in vitro. In both cases, the anti-Env antibodies colocalize with HIV-1 virions, and transcytose across epithelial/endothelial cells. However, we show that bNAbs can inhibit the infectivity of transcytosed virions in both models (Lorin et al., Mucosal Immunology 2017), (Lorin et al., mBio 2020). Second, the development of bNAbs-like IgA+ memory B cells in response to HIV-1 infection has been poorly explored. Hence, we have characterized at the molecular, structural and functional level in vitro and in vivo novel anti-HIV-1 IgA bNAbs isolated from a "viremic controller". Three lineages of bNAbs, 7-269 IgA, 7-176 IgG/IgA and 7-155 IgG were isolated from the HIV-1 controller. Epitope mapping and structural analyses show that these bNAbs from the three clonotypes target the N332 glycan patch associated with the V3 loop, with a partial overlap of their respective epitopes. Their antiviral properties are under evaluation. Finally, we demonstrate the in vivo neutralizing capacity of 7-269 IgA bNAb in a humanized mouse model infected with HIV-1, and then treated with combined antiretroviral therapy. Our investigations demonstrate: (i) that one of the plausible mechanisms by which bNAbs prevent the transmission and dissemination of HIV-1 is that although they do not block virus transcytosis across genital epithelial cells, they neutralize transcytosed virions; (ii) the crucial role of neutralization by anti-HIV-1 antibodies in the protection against HIV-1 brain invasion despite their inability to inhibit transendothelial viral migration; (iii) that like IgG bNAbs, IgA bNAbs likely contribute actively to the antiviral immunity against HIV-1 and may therefore, play a key role in the protection.; Un vaccin efficace contre le VIH-1 devrait induire des anticorps largement neutralisants (bNAbs), actifs contre la plupart des quasi-espèces de VIH-1 qui circulent chez les individus infectés dans le monde. De tels bNAbs sont produits chez 1-2 % des individus infectés. Leur administration passive a montré leur efficacité dans des études précliniques et cliniques. Dans ce travail, nous avons cherché à comprendre les mécanismes d'action par lesquels les bNAbs IgG et IgA sont capables d'interférer sur la transmission et la propagation du VIH-1. Deux problématiques de recherche ont été investiguées : Dans un premier temps, nous avons étudié le rôle des anticorps anti-VIH-1 dans la protection de deux barrières tissulaires d'intérêt dans l'infection par le VIH-1 : l'épithélium du tractus génital féminin et l'endothélium de la barrière hémato-encéphalique. En utilisant des systèmes de transmigration virale à travers des monocouches cellulaires couplés à des analyses de microscopie confocale, nous montrons que bien que l'enveloppe virale n'est pas (dans l'épithélium) ou partiellement requise (dans l'endothélium) pour une transcytose virale optimale, aucun des bNAbs et des anticorps non ou faiblement neutralisants testés ne bloquent le passage du virus à travers ces deux barrières cellulaires in vitro. Dans les deux cas, les complexes anticorps-VIH-1 traversent les cellules par transcytose. Toutefois, les bNAbs inhibent l'infectivité des virions transcytosés dans les deux modèles (Lorin et al., Mucosal Immunology 2017), (Lorin et al., mBio 2020). Le développement potentiel de lymphocytes B mémoires IgA+ de type bNAbs au cours de l'infection à VIH-1 a été très peu exploré. Nous avons donc dans un deuxième temps, caractérisé au niveau moléculaire, structural et fonctionnel in vitro et in vivo, de nouveaux bNAbs anti-VIH-1 de type IgA, isolés d'un « contrôleur virémique ». Trois lignages lymphocytaires de bNAbs, 7-269 IgA, 7-176 IgG/IgA et 7-155 IgG ont été isolés chez ce contrôleur séropositif. La cartographie épitopique et les analyses structurales montrent que les bNAbs des trois clonotypes ciblent un épitope au niveau des N-glycanes associés au supersite N332 à la base de la boucle V3 du trimère, avec un recouvrement partiel de leurs épitopes respectifs. Leurs propriétés antivirales sont en cours d'évaluation. Enfin, nous avons démontré la capacité neutralisante in vivo du bNAb IgA 7-269 dans un modèle de souris humanisées infectées puis traitées par un traitement antirétroviral combiné. Nos investigations démontrent : (i) qu'un des mécanismes probables par lequel les bNAbs préviennent la transmission de l'infection est qu'ils ne bloquent pas le transport du virus à travers les cellules épithéliales génitales mais qu'ils neutralisent les virions transcytosés ; (ii) le rôle clé de la neutralisation par les anticorps anti-VIH-1 dans la protection contre l'invasion virale du système nerveux central ; (iii) que les bNAbs IgA anti-VIH-1 au même titre que les bNAbs IgG pourraient participer de manière efficace à l'immunité anti-VIH-1.

Since the outbreak of the global pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), several clinical aspects of the disease have come into attention. Besides its primary route of infection through the respiratory system, SARS-CoV-2 is known to have neuroinvasive capacity, causing multiple neurological symptoms with increased neuroinflammation and blood–brain barrier (BBB) damage. The viral spike protein disseminates via circulation during infection, and when reaching the brain could possibly cross the BBB, which was demonstrated in mice. Therefore, its medical relevance is of high importance. The aim of this study was to evaluate the barrier penetration of the S1 subunit of spike protein in model systems of human organs highly exposed to the infection. For this purpose, in vitro human BBB and intestinal barrier cell–culture systems were investigated by an optical biosensing method. We found that spike protein crossed the human brain endothelial cell barrier effectively. Additionally, spike protein passage was found in a lower amount for the intestinal barrier cell layer. These observations were corroborated with parallel specific ELISAs. The findings on the BBB model could provide a further basis for studies focusing on the mechanism and consequences of spike protein penetration across the BBB to the brain.

Tricellulin (Tric) is a tight junction protein at tricellular contacts; however its exact structure, function and regulation are unclear. Tric contributes to the paracellular tightening by intercellular trans-interactions between its extracellular loops 2 (ECL2). Consequently, trictide, a peptide derived from the Tric ECL2 was designed as a potential drug enhancer to specifically overcome tissue barriers. This work is aimed to investigate the functional and molecular modulations of tricellular contacts caused by trictide and to understand essential elements and protein interactions of tricellular tight junctions (tTJ) for barrier formation. Trictide increased the passage of ions, small and larger molecules up to 10 kDa in a concentration dependent manner over duration from 16 h to 47 h after application to human colon epithelial cells. Under normal conditions, lipolysis-stimulated lipoprotein receptor (LSR) and Tric localized at tTJs while occludin (Occl), claudin-5 (Cldn5) and claudin-1 (Cldn1) localized at bicellular TJs (bTJ). After trictide treatment, Tric and LSR moved from tTJs to bTJs, and the bTJ proteins Cldn1 and Occl were internalized. Trictide also decreased the transcellular resistance of brain endothelial cells after 14 h and caused enrichment of Cldn5 around the tri-cellular area. Trictide down-regulated Occl, Tric, Cldn1, Cldn5 in epithelial cells and LSR, Occl, Cldn1 in mouse brain endothelial cells. Trictide-initiated opening of the tricellular sealing tube revealed a Tric-free area at the tricellular region as demonstrated by super resolution microscopy. In different cells, cis-interactions of Tric–Tric (tTJs), Tric–Cldn1, Tric–marvelD3, and Occl–Occl (bTJs) were strongly reduced by trictide treatment. In normal brain capillaries of different species, Tric was localized both at bTJs and tTJs while LSR was found exclusively at tTJs. In vivo, trictide did not increase the uptake of small molecules by mouse kidney and liver but tended to enhance brain uptake. Circular dichroism spectroscopy and molecular modelling suggested that trictide consists of 50% β-sheet structure resulting in an elongated conformation. Contrarily, scrambled trictide has a globular shape. Molecular docking models of trictide support the assumption that outward-directed aromatic amino acids are involved in a binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to improve permeation of small molecules. Moreover, after targeting the cellular junctions, a connection has been disclosed between the disturbances of mutual interactions and the resulting redistribution of TJ proteins and functional alterations of the barrier.