Structural and kinetic basis for heightened immunogenicity of T cell vaccines

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR–peptide–MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)–A2 tumor epitope NY–ESO-1157–165–SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine–tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA–A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials

Increased peptide contacts govern high affinity binding of a modified TCR whilst maintaining a native pMHC docking mode

NaturalT cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A�0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3b loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134TCR made increased contacts with theTax peptide compared with the A6wt/A2- Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134TCR CDR3b loop.This peptide-focused enhancedTCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies

Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV-2 infection: a nested, case-control diagnostic accuracy study

Background We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing.Methods We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew’s Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for “viral infection”, “transcriptome”, “biomarker”, and “blood”. We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity.Findings We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27–47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91–0·99), sensitivity 0·84 (0·70–0·93), and specificity 0·95 (0·85–0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91–0·95).Interpretation Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge

Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure

The Omicron, or Pango lineage B.1.1.529, variant of SARS-CoV-2 carries multiple spike mutations with high transmissibility and partial neutralizing antibody (nAb) escape. Vaccinated individuals show protection from severe disease, often attributed to primed cellular immunity. We investigated T and B cell immunity against B.1.1.529 in triple mRNA vaccinated healthcare workers (HCW) with different SARS-CoV-2 infection histories. B and T cell immunity against previous variants of concern was enhanced in triple vaccinated individuals, but magnitude of T and B cell responses against B.1.1.529 spike protein was reduced. Immune imprinting by infection with the earlier B.1.1.7 (Alpha) variant resulted in less durable binding antibody against B.1.1.529. Previously infection-naïve HCW who became infected during the B.1.1.529 wave showed enhanced immunity against earlier variants, but reduced nAb potency and T cell responses against B.1.1.529 itself. Previous Wuhan Hu-1 infection abrogated T cell recognition and any enhanced cross-reactive neutralizing immunity on infection with B.1.1.529

Quantitative, multiplexed, targeted proteomics for ascertaining variant specific SARS-CoV-2 antibody response

Determining the protection an individual has to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VoCs) is crucial for future immune surveillance, vaccine development, and understanding of the changing immune response. We devised an informative assay to current ELISA-based serology using multiplexed, baited, targeted proteomics for direct detection of multiple proteins in the SARS-CoV-2 anti-spike antibody immunocomplex. Serum from individuals collected after infection or first- and second-dose vaccination demonstrates this approach and shows concordance with existing serology and neutralization. Our assays show altered responses of both immunoglobulins and complement to the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.1) VoCs and a reduced response to Omicron (B1.1.1529). We were able to identify individuals who had prior infection, and observed that C1q is closely associated with IgG1 (r > 0.82) and may better reflect neutralization to VoCs. Analyzing additional immunoproteins beyond immunoglobulin (Ig) G, provides important information about our understanding of the response to infection and vaccination

T cell receptor engagement of peptide-major histocompatibility complex class I does not modify CD8 binding

Activation of cytotoxic T cells is initiated by engagement of the T-cell receptor (TCR) with peptide-major histocompatibility class I complexes (pMHCI). The CD8 co-receptor also binds to pMHCI, but at a distinct site, and allows the potential for tripartite TCR/pMHCI/CD8 interactions, which can increase T cell antigen sensitivity. There has been a substantial interest in the effect of the pMHCI/CD8 interaction upon TCR/pMHCI engagement, and several conflicting studies have examined this event, using the soluble extracellular domains of CD8 and the TCR, by surface plasmon resonance. However, the evidence to date suggests that the TCR engages cognate pMHCI before CD8 recruitment, so the question of whether TCR engagement alters CD8 binding is likely to be more relevant to the biological order of T cell antigen encounter. Here, we have examined the binding of CD8 to several variants of the HLA A2-restricted telomerase(540-548) antigen (ILAKFLHWL) and the HLA A2-restricted NY-ESO-1(157-165) antigen (SLLMWITQC) that bind to their cognate TCRs with distinct affinities and kinetics. These interactions represent a range of agonists that exhibit different CD8 dependency for activation of their respective T cells. By using engineered affinity enhanced TCRs to these ligands, which have extended off-rates of approximately 1h compared to seconds for the wildtype TCRs, we have examined pMHCI/CD8 binding before and during TCR-engagement. Here we show that the binding of the extracellular domain of the TCR to pMHCI does not transmit structural changes to the pMHCI-CD8 binding site that would alter the subsequent pMHCI/CD8 interaction