A universal MHCII technology platform to characterize antigen-specific CD4+ T cells.

CD4+ T cells are critical to the immune system and perform multiple functions; therefore, their identification and characterization are crucial to better understanding the immune system in both health and disease states. However, current methods rarely preserve their ex vivo phenotype, thus limiting our understanding of their in vivo functions. Here we introduce a flexible, rapid, and robust platform for ex vivo CD4+ T cell identification. By combining MHCII allele purification, allele-independent peptide loading, and multiplexed flow cytometry technologies, we can enable high-throughput personalized CD4+ T cell identification, immunophenotyping, and sorting. Using this platform in combination with single-cell sorting and multimodal analyses, we identified and characterized antigen-specific CD4+ T cells relevant to COVID-19 and cancer neoantigen immunotherapy. Overall, our platform can be used to detect and characterize CD4+ T cells across multiple diseases, with potential to guide CD4+ T cell epitope design for any disease-specific immunization strategy.

Immunogenicity and safety of BNT162b2 mRNA vaccine in Chinese adults: A phase 2 randomised clinical trial.

Background: BNT162b2, an mRNA vaccine against COVID-19, is being utilised worldwide, but immunogenicity and safety data in Chinese individuals are limited. Methods: This phase 2, randomised, double-blind, placebo-controlled trial included healthy or medically stable individuals aged 18-85 years enrolled at two clinical sites in China. Participants were stratified by age (≤55 or >55 years) and randomly assigned (3:1) by an independent randomisation professional to receive two doses of intramuscular BNT162b2 30 µg or placebo, administered 21 days apart. Study participants, study personnel, investigators, statisticians, and the sponsor's study management team were blinded to treatment assignment. Primary immunogenicity endpoints were the geometric mean titers (GMTs) of neutralising antibodies to live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seroconversion rates (SCR) 1 month after the second dose. Safety assessments included reactogenicity within 14 days of vaccination, adverse events (AEs), and clinical laboratory parameters. Randomised participants who received at least one dose were included in the efficacy and safety analyses on a complete case basis (incomplete/missing data not imputed). Results up to 6 months after the second dose are reported. Findings: Overall, 959 participants (all of Han ethnicity) who were recruited between December 5th, 2020 and January 9th, 2021 received at least one injection (BNT162b2, n=720; placebo, n=239). At 1 month after the second dose, the 50% neutralising antibody GMT was 294.4 (95% CI; 281.1-308.4) in the BNT162b2 group and 5.0 (95% CI; 5.0-5.0) in the placebo group. SCRs were 99.7% (95% CI; 99.0%-100.0%) and 0% (95% CI; 0.0%-1.5%), respectively (p<0.0001 vs placebo). Although the GMT of neutralising antibodies in the BNT162b2 group was greatly reduced at 6 months after the second dose, the SCR still remained at 58.8%. BNT162b2-elicited sera neutralised SARS-CoV-2 variants of concern. T-cell responses were detected in 58/73 (79.5%) BNT162b2 recipients. Reactogenicity was mild or moderate in severity and resolved within a few days after onset. Unsolicited AEs were uncommon at 1 month following vaccine administration, and there were no vaccine-related serious AEs at 1 month or 6 months after the second dose. Interpretation: BNT162b2 vaccination induced a robust immune response with acceptable tolerability in Han Chinese adults. However, follow-up duration was relatively short and COVID-19 rates were not assessed. Safety data collection is continuing until 12 months after the second dose. Funding: BioNTech - sponsored the trial. Shanghai Fosun Pharmaceutical Development Inc. (Fosun Pharma) - conducted the trial, funded medical writing. ClinicalTrialsgov registration number: NCT04649021. Trial status: Completed.

Personalized neoantigen vaccine NEO-PV-01 with chemotherapy and anti-PD-1 as first-line treatment for non-squamous non-small cell lung cancer.

Neoantigens arising from mutations in tumor DNA provide targets for immune-based therapy. Here, we report the clinical and immune data from a Phase Ib clinical trial of a personalized neoantigen-vaccine NEO-PV-01 in combination with pemetrexed, carboplatin, and pembrolizumab as first-line therapy for advanced non-squamous non-small cell lung cancer (NSCLC). This analysis of 38 patients treated with the regimen demonstrated no treatment-related serious adverse events. Multiple parameters including baseline tumor immune infiltration and on-treatment circulating tumor DNA levels were highly correlated with clinical response. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination. Epitope spread to non-vaccinating neoantigens, including responses to KRAS G12C and G12V mutations, were detected post-vaccination. Neoantigen-specific CD4+ T cells generated post-vaccination revealed effector and cytotoxic phenotypes with increased CD4+ T cell infiltration in the post-vaccine tumor biopsy. Collectively, these data support the safety and immunogenicity of this regimen in advanced non-squamous NSCLC.

Combined TCR Repertoire Profiles and Blood Cell Phenotypes Predict Melanoma Patient Response to Personalized Neoantigen Therapy plus Anti-PD-1.

T cells use highly diverse receptors (TCRs) to identify tumor cells presenting neoantigens arising from genetic mutations and establish anti-tumor activity. Immunotherapy harnessing neoantigen-specific T cells to target tumors has emerged as a promising clinical approach. To assess whether a comprehensive peripheral mononuclear blood cell analysis predicts responses to a personalized neoantigen cancer vaccine combined with anti-PD-1 therapy, we characterize the TCR repertoires and T and B cell frequencies in 21 patients with metastatic melanoma who received this regimen. TCR-α/ß-chain sequencing reveals that prolonged progression-free survival (PFS) is strongly associated with increased clonal baseline TCR repertoires and longitudinal repertoire stability. Furthermore, the frequencies of antigen-experienced T and B cells in the peripheral blood correlate with repertoire characteristics. Analysis of these baseline immune features enables prediction of PFS following treatment. This method offers a pragmatic clinical approach to assess patients' immune state and to direct therapeutic decision making.

A Phase Ib Trial of Personalized Neoantigen Therapy Plus Anti-PD-1 in Patients with Advanced Melanoma, Non-small Cell Lung Cancer, or Bladder Cancer.

Neoantigens arise from mutations in cancer cells and are important targets of T cell-mediated anti-tumor immunity. Here, we report the first open-label, phase Ib clinical trial of a personalized neoantigen-based vaccine, NEO-PV-01, in combination with PD-1 blockade in patients with advanced melanoma, non-small cell lung cancer, or bladder cancer. This analysis of 82 patients demonstrated that the regimen was safe, with no treatment-related serious adverse events observed. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumor and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination. These data support the safety and immunogenicity of this regimen in patients with advanced solid tumors (Clinicaltrials.gov: NCT02897765).

Morphological and phenotypic analyses of the human placenta using whole mount immunofluorescence.

The human placenta performs multiple essential functions required for successful pregnancy. Alterations in the placental vasculature have been implicated in severe complications of pregnancy. Despite the importance of placental vascular function during pregnancy, there are gaps in our knowledge regarding the molecular pathways that control vessel development. Furthermore, there are limited tools available to simultaneously examine the morphology, phenotype, and spatial arrangement of cells within intact placental structures. To overcome these limitations, we developed whole mount immunofluorescence (WMIF) of the human placenta. Morphological analyses using WMIF revealed that blood vessel structures were consistent with an immature, angiogenic morphology in first-trimester placentas and mature, remodeled endothelium at term. To investigate placental expression of factors that control blood vessel development, we utilized WMIF to examine gestation age-specific expression of 1) the receptors for vascular endothelial growth factor (VEGFR-1, VEGFR-2, and VEGFR-3), which are required for placental vascular development in mice, and 2) activated, tyrosine phosphorylated STAT3 (pSTAT3), a transcription factor that mediates VEGFR2 signaling. We detected high levels of VEGFR2, VEGFR3, and pSTAT3 expression in early placental blood vessels that were significantly diminished by term. VEGFR1 was expressed primarily in trophoblast and Hofbauer cells throughout gestation. Based on our collective results, we propose that VEGFR2, VEGFR3, and STAT3 play essential roles in the development of the human placental vasculature. In addition, we anticipate that WMIF will provide a powerful approach for comparing placental morphology and protein expression in normal versus pathological pregnancies and for investigating the effects of environmental factors on placental function.

Coordinate loss of MHC class II expression in the diffuse large B cell lymphoma cell line OCI-Ly2 is due to a novel mutation in RFX-AP.

Loss of major histocompatibility complex class II (MHCII) antigen expression on diffuse large B cell lymphoma (DLBCL) corresponds closely with significant decreases in patient survival. However, the mechanisms accounting for MHCII loss in DLBCL have not been thoroughly characterized to date. In this report, we demonstrate that coordinate loss of MHCII expression in OCI-Ly2 DLBCL cells is associated with an 11-base deletion in the cDNA encoding RFX-AP, one of the subunits of the heterotrimeric regulatory factor X (RFX) that is required for activating MHCII transcription. This deletion results in a frameshift in the RFX-AP protein beginning at amino acid 234 and, therefore, in the loss of C-terminal amino acids that are required for function. Stable transfection of OCI-Ly2 DLBCL cells with an expression vector for wild-type RFX-AP restores MHCII expression, which strongly suggests that the defect in RFX-AP accounts for MHCII loss in these cells.