Project description:Similar with others, our data proved that antigen-specific CD8+ T cells from mice primed with DNA and boosted by VACV were much more sensitive to antigen stimulation than those from DNA-boost. Since the mechanisms of in vivo tuning of antigen sensitivity (also termed functional avidity) is still not defined, we compared this two vaccination regimen at gene expression level. Results provide important information of which genes were selectively activated by VACV boost vaccination. For example, data shows that the expression levels of genes involved in Cancer and Wnt signaling pathways is more higher in DNA prime-VACV boost regimen that DNA prime-DNA boost vaccination. To obtain sufficient of antigen-specific cells for microarray analysis, the OVA-specific CD8+ T cells from OT-1 mice were adoptively transferred into wild type mice and then immunized by DNA and VACV vaccine encoding OVA. Four week later, mice were scarificed and antigen-specific CD8+ T cells were emriched by CD45.1-PE antibody and anti-PE MicroBeads from splenocytes.Total RNA was extracted by the RNeasy Mini Kit (QIAGEN, Germany). Followed by amplification and biotin labeling, the samples were hybridized using Illumina Total Prep RNA Amplification Kit (Ambion, USA). Mouse WG-6v2 Expression BeadChips were used for analysis of transcriptome.
Project description:Similar with others, our data proved that antigen-specific CD8+ T cells from mice primed with DNA and boosted by VACV were much more sensitive to antigen stimulation than those from DNA-boost. Since the mechanisms of in vivo tuning of antigen sensitivity (also termed functional avidity) is still not defined, we compared this two vaccination regimen at gene expression level. Results provide important information of which genes were selectively activated by VACV boost vaccination. For example, data shows that the expression levels of genes involved in Cancer and Wnt signaling pathways is more higher in DNA prime-VACV boost regimen that DNA prime-DNA boost vaccination.
Project description:Introduction: Natural killer T (NKT) cells and CD8+ T cells are key in the immune response against multiple myeloma (MM), a largely incurable blood cancer. Immunization is a promising strategy to activate these T cell populations. To our knowledge, immunization with mRNA and α-galactosyl ceramide (αGC) have not been studied in MM, as knowledge on clinically relevant antigens in preclinical MM models is lacking. Methods: Transcriptomics and immunopeptidomics were used to identify candidate antigens for immunization in 5TMM models. Galsomes, lipid nanoparticles containing antigen mRNA and αGC were used to immunize 5T33MM bearing mice. This treatment was combined with a CD40 agonist. Tumor burden and activation of NKT cells and CD8+ T cells were studied using M-protein electrophoresis, cytospin, flow cytometry and ELISA. Results: Transcriptomics revealed survivin as a candidate antigen. Prime-boost Galsomes therapy targeting survivin significantly reduced M-protein levels despite low survivin-specific T cell responses. Further analysis showed potential T cell fratricide. Immunopeptidomics revealed HSP60, Idiotype, PICALM and EF1A1 as candidate antigens. Prime-boost therapy with Galsomes targeting these antigens reduced MM growth significantly when combined with a CD40 agonist, coinciding with significantly improved antigen presentation, co-stimulation and cytotoxicity of NKT cells and CD8+ T cells. Conclusion: These findings highlight the potential of Galsomes, an mRNA vaccine designed to activate CD8+ T cells and NKT cells, for MM therapy, and emphasize the importance of combinatorial approaches, addressing immune anergy for effective MM immunotherapies.
Project description:Much is known concerning the cellular and molecular basis for CD8+ T memory immune responses. Nevertheless, conditions that selectively support memory generation have remained elusive. Here we show that an immunization regimen that delivers TCR signals through a defined antigenic peptide, inflammatory signals through LPS, and growth and differentiation signals through the IL-2R initially favors antigen-specific CD8+ T cells to rapidly and substantially develop into tissue-residing T effector-memory cells by TCR transgenic OVA-specific OT-I CD8+ T cells. Amplified CD8+ T memory development depends upon a critical frequency of antigen-specific T cells and direct responsiveness to IL-2. A homologous prime-boost immunization protocol with transiently enhanced IL-2R signaling in normal mice led to persistent polyclonal antigen-specific CD8+ T cells that supported protective immunity to Listeria monocytogenes. These results identify a general approach for amplified T memory development that may be useful to optimize vaccines aimed at generating robust cell-mediated immunity. Gene expression analysis was performed for OT-I T cells on day 3 and day 5 after activation with ovalbumin and LPS in vivo with and without treatment with IL-2 using an agonists IL-2/anti-IL-2 complexes (IL2/Jes-6.1) OT-I T cells were purified and adoptively transferred into congenic syngenic mice. 24 hours later mice were immunization with ovalbumin and LPS. 24 hr later some mice received agonist IL2/anti-IL2. 3 and 5 days after immunization, the activated OT-I T cells were purifed by FACS and total RNA was isolated for genome wide expression analysis using Affymetrix Mouse Gene ST1.0 arrays
Project description:Much is known concerning the cellular and molecular basis for CD8+ T memory immune responses. Nevertheless, conditions that selectively support memory generation have remained elusive. Here we show that an immunization regimen that delivers TCR signals through a defined antigenic peptide, inflammatory signals through LPS, and growth and differentiation signals through the IL-2R initially favors antigen-specific CD8+ T cells to rapidly and substantially develop into tissue-residing T effector-memory cells by TCR transgenic OVA-specific OT-I CD8+ T cells. Amplified CD8+ T memory development depends upon a critical frequency of antigen-specific T cells and direct responsiveness to IL-2. A homologous prime-boost immunization protocol with transiently enhanced IL-2R signaling in normal mice led to persistent polyclonal antigen-specific CD8+ T cells that supported protective immunity to Listeria monocytogenes. These results identify a general approach for amplified T memory development that may be useful to optimize vaccines aimed at generating robust cell-mediated immunity. Gene expression analysis was performed for OT-I T cells on day 3 and day 5 after activation with ovalbumin and LPS in vivo with and without treatment with IL-2 using an agonists IL-2/anti-IL-2 complexes (IL2/Jes-6.1)
Project description:The single-cell transcriptional profiling of T cells in the spleens of vaccinated mice revealed that the mRNA-based vaccine significantly promoted CD8+ T cells and memory T cells by prime-boost immunization.
Project description:The generation of CD8+ T-cell memory is an important aim of immunization. While several distinct subsets of CD8+ T-cell memory have been described, the lineage relationships between effector (EFF), effector memory (EM) and central memory (CM) T cells remain contentious. Specifically, there is contradictory experimental evidence to support both the linear (Naive>EFF>EM>CM) and progressive differentiation (Naive>CM>EM>EFF) models. In this study, we applied a systems biology approach to examine global transcriptional relationships between the three major CD8+ T cell subsets arising endogenously as a result of vaccination with three different prime-boost vaccine regimens. Differential gene expression analysis and principle component analysis revealed that central memory cells were more closely related to naive T cells than both effector memory and effector cells. When the transcriptional relationships between subsets were enriched in an unbiased fashion with known global transcriptional changes that result when T-cells repeatedly encounter antigen, our analysis favored a model whereby cumulative antigenic stimulation drives differentiation specifically from Naive > CM > EM > EFF. These findings provide an insight into the lineage relationship between mature CD8+ T-cell subsets and will help in the rational design of vaccines aimed at generating effective immune responses against infections and cancer. Effector (EFF), effector memory (EM), central memory (CM) and naive CD8+ T cells from mice spleen. Memory subset arise endogenously as a result of vaccination with three different prime-boost vaccine regimens: DNA-rAd5, rAd5-rAd5 and rAd5-rLCMV.
Project description:Despite the widespread use of adenovirus, mRNA, and protein-based vaccines during the COVID-19 pandemic, their relative immunological profiles and protective efficacies remain incompletely defined. Here, we compared antigen kinetics, innate and adaptive immune responses, and protective efficacy following Ad5, mRNA, and protein vaccination in mice. Ad5 induced the most sustained antigen expression, but mRNA induced the most potent interferon responses, associated with robust antigen presentation and costimulation. Unlike Ad5 vaccines, which were hindered by pre-existing vector immunity, mRNA vaccines retained efficacy after repeated use. As a single-dose regimen, Ad5 vaccines elicited superior immune responses. However, as a prime-boost regimen, and particularly in Ad5 seropositive mice, mRNA vaccines outperformed the other vaccine platforms. These findings highlight strengths of each vaccine platform and underscore the importance of vaccination context in determining optimal vaccine performance.
Project description:A better understanding of innate responses induced by vaccination is critical for designing optimal vaccines. Here, we studied the diversity and dynamics of the NK cell compartment after prime-boost immunization with the modified vaccinia virus Ankara using cynomolgus macaques as a model. Mass cytometry was used to deeply characterize blood NK cells. The NK cell subphenotype composition was modified by the prime. Certain phenotypic changes induced by the prime were maintained over time and, as a result, the NK cell composition prior to boost differed from that before prime. The key phenotypic signature that distinguished NK cells responding to the boost from those responding to the prime included stronger expression of several cytotoxic, homing, and adhesion molecules, suggesting that NK cells at recall were functionally distinct. Our data reveal potential priming or imprinting of NK cells after the first vaccine injection. This study provides novel insights into prime-boost vaccination protocols that could be used to optimize future vaccines.
Project description:We addressed the question of primed CD8 T cell responsiveness to boost in a Balb/c mouse model of vaccination against gag of HIV-1, namely intramuscular (i.m.) prime with the Chimpanzee adenovector ChAd3-gag and i.m. boost with Modified Virus Ankara MVA-gag. In this setting, boost was more effective at day(d)100 than at d30 post-prime, as evaluated by multi-lymphoid organ assessment of gag-specific CD8 T cell frequency, CD62L-phenotype and in vivo killing activity at d45 post-boost. RNA-sequencing was used to compare memory signature of gag-specific spleen CD8 T cells at d100 post-prime with those at d30.