Caddy, S. | Search bioRxiv

bioRxiv2025

Humanizing a CD28 signaling domain affects CD8 activation, exhaustion and stem-like precursors

CD28 ligation provides critical signals that modulate activated T cell fate. In a human to mouse reverse-engineering approach, a single amino acid substitution adjacent to the C-terminal proline-rich domain created CD28A210P mice with enhanced signaling. CD28A210P mice experienced pro-inflammatory responses to CD28 superagonist antibody, analogous to severe cytokine storm induced in a human clinical trial, with a striking increase of activated CD8 T cells. In acute and chronic viral infections, early activation and expansion of CD28A210P CD8 effector T cells increased, with accelerated exhaustion in chronic infection. Mechanistically, CD28A210P enhanced JunB, IL-2, and inhibitory receptors driven by MEK1/2. Generation of CD28A210P stem-like progenitor (Tpex) cells was enhanced in acute and chronic infections, and further expanded by PD-L1 blockade in chronically-infected mice. Thus, humanized PYAP mice reveal key roles for CD28 signaling strength in CD8 activation, accelerating exhaustion during antigen persistence, while promoting and sustaining Tpex during acute and chronic viral infection. One sentence SummaryA single amino acid substitution adjacent to PYAP to humanize CD28 signaling enhances superagonist response, early CD8 activation and Tpex generation during viral infection while accelerating exhaustion and sustaining Tpex during chronic infection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/642460v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@13fc9f9org.highwire.dtl.DTLVardef@e50762org.highwire.dtl.DTLVardef@47c8eeorg.highwire.dtl.DTLVardef@d892d7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Humanized CD28 PYAPP enhances numbers of CD8 T cell effectors and stem-like precursors during acute viral infection, and accelerates exhaustion while sustaining increased self-renewing Tpex cells that are favored during PD-L1 blockade. C_FIG

immunology↗

bioRxiv2022

Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes

Human rotavirus (RV) vaccines used worldwide have been developed using live attenuated platforms. The recent development of a reverse genetics system for RVs has delivered the possibility of engineering chimeric viruses expressing heterologous peptides from other virus species to generate polyvalent vaccines. We tested the feasibility of this using two approaches. Firstly, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian SA11 RV strain viral protein (VP) 4. Secondly, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C-terminus of non-structural protein (NSP) 3 of the bovine RF strain RV, with or without an intervening T2A peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RF strain. Except for the RBD mutant, all NSP3 mutants delivered endpoint titres and replication kinetics comparable to that of the WT virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants but only RBM mutant showed cross reactivity with SARS-CoV-2 RBD antibody. The tolerability of large peptide insertions in the NSP3 segment highlights the potential for this approach in the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCEWe explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an exemplar. Small SARS-CoV-2 peptide insertion (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titre and replication, thus limiting its use as a potential vaccine expression platform. To test RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids. With a T2A-separated 193 amino acid tag on NSP3, there was little effect on the viral rescue efficiency, titre and replication. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. This is the first report describing epitope tagging of VP4, and of a reverse genetics system for the RF strain. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

microbiology↗