We observe previously unknown interactions between clinically important adenovirus vector capsids, platelet factor 4, and CAR.

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8⁺ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease.

Human adenovirus type 26 uses sialic acid as a primary cellular receptor—structural insights for this phase 3 vaccine vector.

Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are demonstrating promising efficacy as protective platforms against infectious diseases. Significant clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, and receptor usage are currently lacking. Here, we perform structural and biological analysis of the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our findings provide insight into the cell-virus interactions of HAdV-D26/48, with important implications for the design and engineering of optimised Ad-based therapeutics.

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues. Experimental Design: Ad5_NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvβ6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed. Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5_NULL-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5_NULL-A20 efficiently and selectively transduced αvβ6⁺ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5_NULL-A20 following systemic delivery in non–tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 10⁷-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5_NULL-A20–treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5. Conclusions: Oncolytic Ad5_NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215–24. ©2018 AACR.

Presentation of identical HIV-1 peptides by closely related Human Leukocyte Antigen class I (HLAI) molecules can select distinct patterns of escape mutation that have a significant impact on viral fitness and disease progression. The molecular mechanisms by which HLAI micropolymorphisms can induce differential HIV-1 escape patterns within identical peptide epitopes remain unknown. Here, we undertook genetic and structural analyses of two immunodominant HIV-1 peptides, Gag180–188 (TPQDLNTML, TL9-p24) and Nef71–79 (RPQVPLRPM, RM9-Nef) that are among the most highly targeted epitopes in the global HIV-1 epidemic. We show that single polymorphisms between different alleles of the HLA-B7 superfamily can induce a conformational switch in peptide conformation that is associated with differential HLAI-specific escape mutation and immune control. A dominant R71K mutation in the Nef71-79 occurred in those with HLA-B*07:02 but not B*42:01/02 or B*81:01. No structural difference in the HLA-epitope complexes was detected to explain this observation. These data suggest that identical peptides presented through very similar HLAI landscapes are recognized as distinct epitopes and provide a novel structural mechanism for previously observed differential HIV-1 escape and disease progression.