Biology subjects

Mwangi, W.

Publications and source records attributed to Mwangi, W..

9 recordsLinked to original sources

bioRxiv2026

Simplifying principles that underlie the highly complex peptide motif of the promiscuous chicken class I molecule, BF2*21:01

In chickens and humans, classical class I molecules of the major histocompatibility complex (MHC) have a spectrum of correlated properties, including cell surface expression and peptide repertoire. Understanding the impact of these promiscuous generalists and fastidious specialists is of considerable interest. The promiscuous BF2 molecule from the chicken B21 haplotype, BF2*21:01, binds a wide range of peptides by remodelling the peptide-binding site, allowing co-variation of the anchor residues at peptide positions P2 and Pc-2, and binding of an anchor residue at Pc. Using in vitro refolding assays with peptides and peptide libraries, determining thermostability and crystal structures, and analysing a chicken B21 cell line by immunopeptidomics, we found that BF2*21:01 will accommodate many possible combinations at P2 and Pc-2, as well as several hydrophobic amino acids at Pc. However, marked preferences for particular peptide lengths, particular amino acids at the three anchor residues, and particular combinations of amino acids at P2 and Pc-2 as well as at P3 and Pc-3 together lead to high frequencies of major peptides while still allowing the possibility of presenting a wide peptide repertoire. These simplifying principles may eventually allow predictions of pathogen peptides with stable binding for this iconic promiscuous class I molecule, as well as providing the data for more sophisticated peptide prediction methods.

immunology↗

bioRxiv2024

HIV envelope trimers and gp120 as immunogens to induce broadly neutralizing antibodies in cows

The study of immunogens capable of eliciting broadly neutralizing antibodies (bnAbs) is crucial for the development of an HIV vaccine. To date, only cows, making use of their ultralong CDRH3 loops, have reliably elicited bnAbs following immunization with HIV Envelope trimers. Antibody responses to the CD4 binding site have been readily elicited by immunization of cows with a stabilized Env trimer of the BG505 strain and, with more difficulty, to the V2-apex region of Env with a cocktail of trimers. Here, we sought to determine whether the BG505 Env trimer could be engineered to generate new bnAb specificities in cows. Since the cow CD4 binding site bnAbs bind to monomeric BG505 gp120, we also sought to determine whether gp120 immunization alone might be sufficient to induce bnAbs. We found that engineering the CD4 binding site by mutation of a key binding residue of BG505 HIV Env resulted in a reduced bnAb response that took more immunizations to develop. Monoclonal antibodies isolated from one animal were directed to the V2-apex, suggesting a re-focusing of the bnAb response. Immunization with monomeric BG505 g120 generated no serum bnAb responses, indicating that the ultralong CDRH3 bnAbs are only elicited in the context of the trimer in the absence of many other less restrictive epitopes presented on monomeric gp120. The results support the notion of a hierarchy of epitopes on HIV Env and suggest that, even with the presence in the cow repertoire of ultralong CDRH3s, bnAb epitopes are relatively disfavored.

immunology↗

bioRxiv2024

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response. Author SummaryThe elicitation of epitope-specific broadly neutralizing antibodies is highly desirable for an HIV vaccine as bnAbs can prevent HIV infection in robust animal challenge models and humans, but to date, cows are the only model shown to reliably produce HIV bnAb responses on Envelope (Env) immunization. These responses involve Abs with ultralong CDRH3s and are all directed to a single site, the CD4 binding site. To determine whether this is a unique phenomenon or whether cow antibodies can target further bnAb sites on Env, we employed an immunization protocol that generated cow bnAbs to a second site, the V2-apex. We conclude that ultralong CDRH3s are well adapted to penetrate the glycan shield of HIV Env and recognize conserved regions and may constitute protein units, either in the context of antibodies or in other engineered proteins, that could be deployed as anti-HIV reagents.

immunology↗

bioRxiv2024

Ability of mycobacterial pathogens to establish a persistent infection is lost by deletion of a single gene, rel, regulator of the stringent response

Studies in a mouse model revealed Mycobacterium tuberculosis (Mtb) with a deletion of rel, regulator of the stringent response, could not establish a persistent infection. Studies in cattle with a Mycobacterium. a. paratuberculosis rel deletion mutant revealed inability to establish a persistent infection was associated with development of CD8 cytotoxic T cells (CTL) that kill intracellular bacteria. Further comparative studies ex vivo with Mbv Calmette-Guerin (BCG) and a BCG rel deletion mutant revealed no clear difference in development of CTL in vitro. As reported, a study of the recall response was conducted with cattle vaccinated with either BCG or with BCGrel, to determine if information could be obtained that would show how gene products under control of rel interfere with the CTL response to mycobacterial pathogens in vivo. The study revealed the CTL response elicited by vaccination with BCG was impaired, in comparison with the response elicited by BCGrel. Comparative analysis of the recall response ex vivo revealed the functional impairment was not associated with the timing of appearance of the recall response, expression of IFN-{gamma}, TNF-, IL-17, or IL-22, or molecules that mediate intracellular killing. Further studies are needed to determine how CD8 CTL functional activity is modulated in vivo by gene products regulated by rel.

immunology↗

bioRxiv2022

Minor differences in peptide presentation between chicken MHC class I molecules can explain differences in disease susceptibility

The highly polymorphic classical major histocompatibility complexes (MHCs) can confer resistance or susceptibility to diseases. The chicken MHC is known to confer decisive resistance or susceptibility to various economically-important pathogens, including the iconic oncogenic herpesvirus that causes Mareks disease (MD). Only one classical class I gene, BF2, is expressed at a high level in chickens, so it was relatively easy to discern a hierarchy from well-expressed thermostable fastidious specialist alleles to promiscuous generalist alleles that are less stable and expressed less on the cell surface. The BF2*1901 is more highly expressed and more thermostable than the closely-related BF2*1501, but the data for peptide repertoire available did not obviously correlate as expected. Here, we confirm for newly-developed chicken lines that the chicken haplotype B15 confers resistance to MD compared to B19. Using gas phase sequencing of peptide pools, and using immunopeptidomics involving mass spectroscopy, we find that the BF2*1901 binds a greater variety of amino acids in some anchor positions than BF2*1501. However, by X-ray crystallography, we find that the peptide-binding groove of BF2*1901 is narrower and shallower. Though the self-peptides bound to BF2*1901 may appear more various than those of BF2*1501, the structures show that the wider and deeper peptide-binding groove of BF2*1501 allows it to tightly accept many more peptides overall, correlating with the expected hierarchies for expression level, thermostability and MD resistance. Moreover, the 2 helix of BF2*1901 is higher than BF2*1501, potentially reducing the number of T cell clones that can recognize this fastidious class I molecule. IMPORTANCEDisease susceptibility mechanism is complicated, but chicken infection of Mareks disease (MD) is one of ideal models, considering the only one highly expressed classical MHC class I, BF2. The different susceptibility of the two close alleles BF2*1901 and BF2*1501 with minor difference of expression and thermostability is still unfathomed. Here, we confirm B15 chicken confers resistance to MD compared to B19. But the BF2*1901 binds a broader variety of anchoring peptides than BF2*1501. This mystery was solved by the structural determination of the two molecules with one similar peptide. The wider and deeper peptide-binding groove of BF2*1501 allows it to tightly accept many more peptides overall, which is concordant to the expected hierarchies for expression level, thermostability and MD resistance.

immunology↗

bioRxiv2022

Cattle T Cell Phenotyping by an 8-Colour, 10-Parameter Panel

Purpose and appropriate sample typesThis multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional ({gamma}{delta}-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naive (TNaive), central memory (TCM), effector memory (TEM) and terminal effector (TTE) T cells. Activated cattle T cells (TAV) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-colour, 10-parameter flow cytometry panel simultaneously identifies cattle TNaive, TAV, TCM, TEM, TTE and {gamma}{delta}-TCR cells. This panel will improve our ability to examine T cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimised for other bovid species as many of these reagents are likely to cross react.

immunology↗

bioRxiv2021

Optimized Multicolour Immunofluorescence Panel for Cattle B Cell Phenotyping by an 8-Colour, 10-Parameter Panel

This 8-colour, 10-parameter panel has been optimised to distinguish between functionally distinct subsets of cattle B cells in both fresh and cryopreserved peripheral blood mononuclear cells (PBMCs). Existing characterised antibodies against cell surface molecules (immunoglobulin light chain (S-Ig(L)), CD20, CD21, CD40, CD71 and CD138) enabled the discrimination of 24 unique populations within the B cell population. This allows the identification of five putative functionally distinct B cell subsets critical to infection and vaccination responses; 1) naive B cells (BNaive), 2) regulatory B cells (BReg), 3) memory B cells (BMem), 4) plasmablasts (PB) and 5) plasma cells (PC). Although CD3 and CD8 can be included as an additional dump channel, it does not significantly improve the panels ability to separate "Classical" B cells. This panel will promote better characterisation and tracking of B cell responses in cattle as well as other bovid species as the reagents are likely to cross react.

immunology↗

bioRxiv2020

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

immunology↗

bioRxiv2018

Identification of a Novel Neutralizing and Two Non-Neutralizing Epitopes on Epstein-Barr Virus gp350 Protein

Prevention of Epstein-Barr virus (EBV) primary infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). To date, eight gp350 epitopes have been identified, but only one has elicited nAbs. In this study, we generated 23 hybridomas that produced anti-gp350 antibodies. We compared the candidate anti-gp350 antibodies to nAb-72A1 by: (1) testing their ability to detect gp350 using ELISA, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using RepliTope peptide microarrays. Eight antibodies recognized both denatured and non-denatured gp350, whereas five failed to react with denatured gp350 but recognized native gp350, suggesting they recognized conformational epitope(s). Sequence analysis of the heavy and light chain variable regions of the hybridomas identified 15 as mAbs with novel CDR regions unique from those of nAb-72A1. Seven of the new mAbs neutralized EBV in vitro, with HB20 and HB17 reducing EBV infection by 40% and >60%, and >30% and 80%, at 10 g/ml and 50 g/ml, respectively. Epitope mapping identified nine epitopes and defined their core residues, including two unique immunodominant epitopes, 253TPIPGTGYAYSLRLTPRPVSRFL253 and 875LLLLVMADCAFRRNLSTSHTYTTPPY899, and a novel nAb epitope 381GAFASNRTFDIT392. This study provides comprehensive in vitro mapping of the exact residues defining nine epitopes of EBV gp350. Our findings will inform novel strategies to design optimal EBV vaccines capable of conferring broader protection against the virus.\n\nImportanceNeutralizing antibodies (nAbs) directed against Epstein-Barr virus envelope glycoprotein gp350/220 (gp350) are generated in humans upon infection or immunization, and are thought to prevent neonatal infection. However, clinical use of exogenous nAbs (passive immunization) is limited to a single study using the only well-characterized nAb, 72A1. The gp350 ectodomain contains at least eight unique B-cell binding epitopes; two of these epitopes are recognized by nAb-72A1. The exact amino acid residues of the other six epitopes and their role in generating nAbs has not been elucidated. We used our 15 newly generated and fully characterized monoclonal antibodies and a peptide-overlapping RepliTope array to provide a comprehensive map of the core amino acid residues that define epitopes of gp350 and to understand their role in generating nAbs. These results will inform design of better-targeted gp350 peptide vaccines that contain only protective epitopes, which will focus the B-cell response to produce predominantly nAbs.

immunology↗