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: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: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.
Project description:The goal of an effective AIDS vaccine is to generate immunity that will prevent HIV-1 acquisition. Despite limited progress towards this goal, renewed optimism has followed the recent success of the RV144 vaccine trial in Thailand. However, the lack of complete protection in this trial suggests that breakthroughs, where infection occurs despite adequate vaccination, will be a reality for many vaccine candidates. We previously reported that neutralizing antibodies elicited by DNA prime/rAd5 boost vaccination with SIVmac239 Gag/Pol and Env provided protection against pathogenic SIVsmE660 acquisition after repeated mucosal challenge. Here, we report that SIV-specific CD8+ T cells elicited by that vaccine lowered both peak and set-point viral loads in macaques that became infected despite vaccination. These SIV-specific CD8+ T cells showed strong virus inhibitory activity (VIA) and displayed an effector memory (EM) phenotype. VIA correlated with high levels of CD107a mobilization and perforin expression in SIV-specific CD8+ T cells. Remarkably, both the frequency and the number of Gag CM9-specific public clonotypes were strongly correlated with VIA mediated by EM CD8+ T cells. The ability to elicit such virus-specific EM CD8+ T cells might contribute substantially to an efficacious HIV/AIDS vaccine, even after breakthrough infection. Gag CM9-specific EM CD8+ T cells (CD28 low CD95 high tetramer+) from SIV-negative macaques at 12 wks post-DNA/rAd5 immunization were sorted by flow cytometry for microarray studies. RNA samples from strong VIA animals with (n=3) or without (n=6) CM9 peptide stimulation, along with CM9 peptide stimulated samples from weak VIA animals (n=2) were prepared using the Illumina beads station assay and hybridized to the Illumina HumanHT-12 version 4 Expression BeadChip.
Project description:A safe and effective vaccine for the prevention of human immunodeficiency virus type 1 (HIV-1) infection is a global priority. Tthe efficacy of a DNA prime–recombinant adenovirus type 5 boost (DNA/rAd5) vaccine regimen was testd in persons at increased risk for HIV-1 infection in the United States. The DNA/rAd5 vaccine regimen did not reduce either the rate of HIV-1 acquisition or the viral-load set point in the population studied. System biology analysis, including RNA-sequencing, was used to identify markers of risk of HIV-1 acquisition.