Single domain antibody multimers confer protection against rabies infection

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90–95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection

Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis

Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5′-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein–protein interaction with the cellular translation machinery

Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

Background: Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies. Results: Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. Conclusion: The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but als

Etude d'infections persistantes sur culture cellulaire (BHK21) avec le virus rabique

SIGLECNRS T 56820 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Prise en soins des troubles alimentaires pédiatriques‎ : création d’un protocole d’accompagnement parental axé sur l’alimentation autonome ; étude de cas multiples

Autonomous feeding is an approach to food diversification commonly used in children without difficulties; it is a source of many benefits and respects the developmental trajectory of the child regarding his or her skills. The objective of our work is based on the creation of a therapeutic protocol based on autonomous feeding to take care of patients with pediatric eating disorders in a context of parental support. Thus, our desire is to propose an additional intervention tool in this recent field of competence of speech therapists. We tested this protocol with 4 patients aged between 6 and 12 months to quantitatively and qualitatively measure the first effects of such an intervention tool. A decrease in the impact of the disorder on family dynamics and an increase in the food inventory were found for all patients. Moreover, the majority of speech therapists and parents appreciated this protocol. Thus, these first results are encouraging and lead us to consider independent feeding as a relevant therapeutic tool in the care of these patients. The limitations of this study lie in the small sample to test our protocol, which does not allow a generalization of our results. A new study with a larger cohort and a control group would allow us to obtain more robust and significant statistical data. On the one hand, the protocol respects the current recommendations of the HAS since it is part of an early intervention approach, fully integrating the family, and on the other hand, it encourages the rapid proposal of adapted pieces favoring an optimal oro-motor development. Finally, parental support improves parental skills, which is conducive to the child's progress.Prise en soins des troubles alimentaires pédiatriques : création d'un protocole d'accompagnement parental axé sur l'alimentation autonome ; étude de cas multiples Présenté et soutenu par Léna TUFFEREAU Mémoire professionnel Mémoire codirigé par Mathilde Menu et Caroline Dubois-Levasseur, orthophonistes Mémoire soutenu publiquement le 22 juin 2023 devant le jury composé de Mathilde MENU Orthophoniste Codirectrice de mémoire, membre du jury Caroline DUBOIS LEVASSEUR Orthophoniste Codirectrice de mémoire, membre du jury Gaëlle DE LA VILLEON Pédiatre Présidente du jury Pauline BRUNEL DUFOUR Orthophoniste Membre du jury Audrey AMAND Orthophoniste Membre du jury « 5 fruits et légumes par jour, ils me font marrer... Moi, à la troisième pastèque, je cale. » Pierre Desproge

Infections persistantes in vitro par le virus Sendai : etablissement de l'infection par un melange de particules standart et defectives interferentes sur cellules BHK21

CNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

Mutations Conferring Resistance to Neutralization by a Soluble Form of the Neurotrophin Receptor (p75NTR) Map outside of the Known Antigenic Sites of the Rabies Virus Glycoprotein

The neurotrophin receptor (p75NTR) serves as a receptor for rabies virus (RV). We expressed and purified a soluble chimera consisting of the p75NTR ectodomain fused to the human immunoglobulin G1 (IgG1) Fc fragment (p75-Fc). Although p75-Fc interacts with RV, the infectivity of RV did not decrease significantly when it was incubated in the presence of the soluble receptor alone. However, when it was subsequently incubated with an antihuman IgG directed against the Fc fragment of p75-Fc, the infectivity of RV was significantly lowered (>90%), whereas incubation with antihuman IgG alone had no effect. We then selected eight independent RV mutants that were not neutralized by p75-Fc and antihuman IgG (srr [soluble receptor resistant] mutants). Each mutant carried a single mutation in the glycoprotein gene leading to one amino acid substitution in the protein. A total of four different substitutions were found. Two of the mutations were located at position 318 (phenylalanine replaced by a serine or a valine residue), and two were located at position 352 (histidine replaced by a tyrosine or an arginine residue). All of the mutations prevented the interaction with p75NTR as either a soluble or a membrane-anchored form. Two mutants (F318S) and (H352R) resulted in the formation of small plaques on BSR cells, probably due to the slower maturation of the glycoprotein. Immunoprecipitation, immunofluorescence, and neutralization assays showed that the four mutated glycoproteins still interacted with representative anti-RV glycoprotein monoclonal antibodies (MAbs), indicating that p75NTR binds outside of the known RV glycoprotein antigenic sites