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Promotion of CTL epitope presentation by a nanoparticle with environment-responsive stability and phagolysosomal escape capacity.

ABSTRACT: Vaccines that induce cytotoxic T lymphocyte (CTL)-mediated immune responses constitute an important class of medical tools to fend off diseases like infections and malignancy. Epitope peptides, as a format of CTL vaccines, are being tested preclinically and clinically. To elicit CTL responses, epitope vaccines go through an epitope presentation pathway in dendritic cells (DCs) that has multiple bottleneck steps and hence is inefficient. Here, we report the development of a strategy to overcome one of these barriers, phagolysosomal escape in DCs. First, we furnished a previously established carrier-an immune-tolerant elastin-like polypeptide nanoparticle (iTEP NP)-with the peptides that are derived from the DNA polymerase of herpes simplex virus 1 (Pol peptides). Pol peptides were reported to facilitate phagolysosomal escape. In this study, while we found that Pol peptides promoted the CTL epitope presentation; we also discovered Pol peptides disrupted the formation of the iTEP NP. Thus, we engineered a series of new iTEPs and identified several iTEPs that could accommodate Pol peptides and maintain their NP structure at the same time. We next optimized one of these NPs so that its stability is responsive to its redox environment. This environment-responsive NP further strengthened the CTL epitope presentation and CTL responses. Lastly, we revealed how this NP and Pol peptides utilized biological cues of phagolysosomes to realize phagolysosomal escape and epitope release. In summary, we developed iTEP NP carriers with a new phagolysosomal escape function. These carriers, with their priorly incorporated functions, resolve three bottleneck issues in the CTL epitope presentation pathway: vaccine uptake, phagolysosomal escape, and epitope release.

SUBMITTER: Dong S

PROVIDER: S-EPMC8729261 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

Project description:As human immunodeficiency virus (HIV) begins to replicate within hosts, immune responses are elicited against it. Escape mutations in viral epitopes-immunogenic peptide parts presented on the surface of infected cells-allow HIV to partially evade these responses, and thus rapidly go to fixation. The faster they go to fixation, i.e., the higher their escape rate, the larger the selective pressure exerted by the immune system is assumed to be. This relation underpins the rationale for using escapes to assess the strength of immune responses. However, escape rate estimates are often obtained by employing an aggregation procedure, where several mutations that affect the same epitope are aggregated into a single, composite epitope mutation. The aggregation procedure thus rests upon the assumption that all within-epitope mutations have indistinguishable effects on immune recognition. In this study, we investigate how violation of this assumption affects escape rate estimates. To this end, we extend a previously developed simulation model of HIV that accounts for mutation, selection, and recombination to include different distributions of fitness effects (DFEs) and inter-mutational genomic distances. We use this discrete time Wright-Fisher based model to simulate early within-host evolution of HIV for DFEs and apply standard estimation methods to infer the escape rates. We then compare true with estimated escape rate values. We also compare escape rate values obtained by applying the aggregation procedure with values estimated without use of that procedure. We find that across the DFEs analyzed, the aggregation procedure alters the detectability of escape mutations: large-effect mutations are overrepresented while small-effect mutations are concealed. The effect of the aggregation procedure is similar to extracting the largest-effect mutation appearing within an epitope. Furthermore, the more pronounced the over-exponential decay of the DFEs, the more severely true escape rates are underestimated. We conclude that the aggregation procedure has two main consequences. On the one hand, it leads to a misrepresentation of the DFE of fixed mutations. On the other hand, it conceals within-epitope interactions that may generate irregularities in mutation frequency trajectories that are thus left unexplained.

Project description:Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two cocirculating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to a decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In-depth study of unique changes in epitope D indicated that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands.IMPORTANCE Human norovirus causes ?20% of all acute gastroenteritis and ?200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30?years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time, resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity.

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Promotion of CTL epitope presentation by a nanoparticle with environment-responsive stability and phagolysosomal escape capacity.

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