A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

Molecular Epidemiology and Evolution of Human Respiratory Syncytial Virus and Human Metapneumovirus

Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are ubiquitous respiratory pathogens of the Pneumovirinae subfamily of the Paramyxoviridae. Two major surface antigens are expressed by both viruses; the highly conserved fusion (F) protein, and the extremely diverse attachment (G) glycoprotein. Both viruses comprise two genetic groups, A and B. Circulation frequencies of the two genetic groups fluctuate for both viruses, giving rise to frequently observed switching of the predominantly circulating group. Nucleotide sequence data for the F and G gene regions of HRSV and HMPV variants from the UK, the Netherlands, Bangkok and data available from Genbank were used to identify clades of both viruses. Several contemporary circulating clades of HRSV and HMPV were identified by phylogenetic reconstructions. The molecular epidemiology and evolutionary dynamics of clades were modelled in parallel. Times of origin were determined and positively selected sites were identified. Sustained circulation of contemporary clades of both viruses for decades and their global dissemination demonstrated that switching of the predominant genetic group did not arise through the emergence of novel lineages each respiratory season, but through the fluctuating circulation frequencies of pre-existing lineages which undergo proliferative and eclipse phases. An abundance of sites were identified as positively selected within the G protein but not the F protein of both viruses. For HRSV, these were discordant with previously identified residues under selection, suggesting the virus can evade immune responses by generating diversity at multiple sites within linear epitopes. For both viruses, different sites were identified as positively selected between genetic groups

Secondary-structure prediction revisited: Theoretical β-sheet propensity and coil propensity represent structures of amyloids and aid in elucidating phenomena involved in interspecies transmission of prions

Prions are unique infectious agents, consisting solely of abnormally-folded prion protein (PrPSc). However, they possess virus-like features, including strain diversity, the ability to adapt to new hosts and to be altered evolutionarily. Because prions lack genetic material (DNA and RNA), these biological phenomena have been attributed to the structural properties of PrPSc. Therefore, many structural models of the structure of PrPSchave been proposed based on the limited structural information available, regardless of the incompatibility with high-resolution structural analysis. Recently hypothesized models consist solely of β- sheets and intervening loops/kinks; i.e. parallel in-register β-sheet and β-solenoid models. Owing to the relative simplicity of these structural models of PrPSc, we hypothesized that numerical conversion of the primary structures with a relevant algorithm would enable quantitative comparison between PrPs of distinct primary structures. We therefore used the theoretical values of β-sheet (Pβ) and random-coil (Pc) propensity calculated by secondary structure prediction with a neural network, to analyze interspecies transmission of prions. By reviewing experiments in the literature, we ascertained the biological relevance of Pβ and Pc and found that these classical parameters surprisingly carry substantial information of amyloid structures. We also demonstrated how these parameters could aid in interpreting and explaining phenomena in interspecies transmissions. Our approach can lead to the development of a versatile tool for investigating not only prions but also other amyloids

Development and validation of an epitope-blocking ELISA using an anti-haemagglutinin monoclonal antibody for specific detection of antibodies in sheep and goat sera directed against peste des petits ruminants virus

International audiencePeste des petits ruminants (PPR) is a contagious and economically important disease affecting production of small ruminants (i.e., sheep and goats). Taking into consideration the lessons learnt from the Global Rinderpest Eradication Programme (GREP), PPR is now targeted by the international veterinary community as the next animal disease to be eradicated. To support the African continental programme for the control of PPR, the Pan African Veterinary Vaccine Centre of the African Union (AU-PANVAC) is developing diagnostics tools. Here, we describe the development of a blocking enzyme-linked immunosorbent assay (bELISA) that allows testing of a large number of samples for specific detection of antibodies directed against PPR virus in sheep and goat sera. The PPR bELISA uses an anti-haemagglutinin (H) monoclonal antibody (MAb) as a competitor antibody, and tests results are interpreted using the percentage of inhibition (PI) of MAb binding generated by the serum sample. PI values below or equal to 18% (PI ae 18%) are negative, PI values greater than or equal to 25% (PI ae 25%) are positive, and PI values greater than 18% and below 25% are doubtful. The diagnostic specificity (DSp) and diagnostic sensitivity (DSe) were found to be 100% and 93.74%, respectively. The H-based PPR-bELISA showed good correlation with the virus neutralization test (VNT), the gold standard test, with a kappa value of 0.947. The H-based PPR-bELISA is more specific than the commercial kit ID ScreenA (R) PPR Competition (N-based PPR-cELISA) from IDvet (France), but the commercial kit is slightly more sensitive than the H-based PPR-bELISA. The validation process also indicated good repeatability and reproducibility of the H-based PPR-bELISA, making this new test a suitable tool for the surveillance and sero-monitoring of the vaccination campaign