1 Exploiting genome biology to understand immune response in health and diseaseSergey Belikov

2 I'm gonna make you an offer…

3 Braga et al. Hum. Mol. Genet. 2016

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5 Single-cell assays retain cellular heterogeneity info that is lost by bulk genomic approaches.Bulk Single-cell Bulk analysis Single-cell analysis Braga et al. Hum. Mol. Genet. 2016

6 Combinatorial indexed ChIP to characterize co-occurrence of histone modifications and trx factor binding 1. Cells are crosslinked 2. Chromatin is sheared and 3. immobilized on specific antibody-coated magnetic beads. 4. Chromatin is indexed by ligation of sequencing adaptors. 5. Chromatin is released from the beads and pooled together with other samples in a single tube. 6. The chromatin pool is subjected to a second chromatin IP step. 7. RNA and proteins are degraded and DNA is reverse crosslinked. 8. ChIPed DNA is purified. 9. DNA is amplified by PCR. 10. Sequencing of Co-ChIP libraries Active Poised co-ChIP

7 Adaptive immunological memory involves gene recombination in B and T lymphocytes.Confers high specificity and pathogen-specific protection (up to decades). “Trained immunity” – innate immune memory that induces enhanced inflammatory and antimicrobial properties in innate immune cells. Results in an increased nonspecific response to infections and improved survival of the host. Netea et al., Science, 2016

8 Initial activation of trx is accompanied by the acquisition of specific chromatin marks.They are only partially lost after elimination of the stimulus. The enhanced epigenetic status - H3K4me1 (poised enhancers) results in a stronger response to secondary stimuli upon rechallenge. X Netea et al., Science, 2016

9 Enhancers and promoters-at considerable distance from the promoter, -can be moved or inverted and still function, -many TF binding sites Promoter: DNA segment that initiates trx of a gene, located near the trx start sites, upstream Enhancers are enriched for H3K4me1, high levels of H3K4me3 predominantly mark promoter... Buecker & Wysocka, Cell, 2012

10 H3K4me1 is associated with both active and poised enhancers, while active enhancers are also marked by H3K27 acetylation. Buecker & Wysocka, Cell, 2012

11 MPhs acquire repertoires of active enhancers that are instructed by the microenvironmental signals specific to given tissue… … which affects the regulatory landscape of a cell via the induction of specific trx factors, leading to the expression of genes involved in the unique functional pathways of each tissue-specific cell type

12 TI leads to adaptive states that protect the host after infectionsTI leads to adaptive states that protect the host after infections. However, TI may result in maladaptive states such as postsepsis immune paralysis or hyperinflammation -> T2D, Alzheimer’s disease Netea et al., Science, 2016

13 Trx memory in the adaptive immune responseWhen stimulated for the first time naive cells udergo clonal expansion. Once antigen is cleared, 90-95% of the antigen-specific effector cells die and surviving cells form a pool of long-lived memory cells

14 Woodworth & Holloway, APCSB, 2016The role of epigenetic regulation in trx memory in the adaptive immune response Woodworth & Holloway, APCSB, 2016

15 Inducible chromatin priming and establishment of trx memory in T cellsPrimed DHSs maintain regions of active chromatin close to inducible genes and enhancers that regulate immune response by facilitating the induction of regulatory elements of previously activated T cells Bevington et al., EMBO J , 2016

16 Cohort studies to find sources of genetic variation between groupsHealthy cohort Disease cohort Cohort studies to find sources of genetic variation between groups SNP in AP-1-binding motif (associated with Crohn disease) disrupts AP-1 binding to an enhancer (active in healthy monocytes). The resulting disruption of the chromatin state leads to altered gene trx and provides the mechanism of disease. Winter et al., Immunology , 2017

17 Molecular mechanisms to enhance DNA vaccine immunogenicity

18 Codon optimality After trx, mRNA can be degraded, stored or used for translation

19 Codon optimality is a powerful determinant of mRNA stabilityEach reporter encodes the same polypeptide sequence but is composed of different codon composition of varying optimality Radhakrishnan et al., Cell 2016

20 The pace of translation can regulate protein folding which frequently starts co‐translationally.

21 However... Codon optimization not always positively correlates with DNA vaccine efficiency Foreign sequences could provide better adjuvant effect via patter recognition receptors

22 DNA backbone: Plasmid vector or...Remove bacterial elements! (inhibit transgene expression) Minicircle DNA (mvDNA) vectors generally biosynthesized in recombinant bacteria ...by in vivo site-specific recombination; this strategy allows the removal of surplus parental plasmid species and mini-plasmids that result from the recombination process ...have minimalistic backbone Advantages Disadvantages A safer and more robust construct Size? Enabling a higher effective dose Enhanced T-cell responseg

23 Choice of promoter Natural Pol II promoters are week!Virus-derived promoters: CMV, SV40 However... ... the potency of viral promoters does not necessarily correlate with DNA vaccine efficacy in vivo, ...viral promoters are downregulated by IFN-g, which is produced as a part of inflammatory reaction triggered by unmethylated CpG sequences present in plasmid backbone (recognized by pattern recognition receptors)

24 Adjuvants (molecular)To upregulate To downregulate Cytokine genes, PRRs, genes coding for signaling proteins (HSP70, IRF) RNAi: Genes that suppress vaccine action (FoxO3- suppressive regulator of T-cell proliferation) RNAi: post-trx gene silencing by destruction of specific mRNAs triggered by ds short hairpin RNA structures

25 mRNA vaccines Ex vivo transfection of dendritic cellsSelf-amplifying Non-amplifying (naked)

26 Proteins can be expressed in Xenopus oocytes via injection of synthetic mRNA(s) into oocyte cytoplasm DNA mRNA GR FoxA1 GR+ mRNA FoxA1 injected - Large size, facilitates microinjection. mRNA directed translation, up to 1pmol protein

27 Dendrimer-RNA nanoparticles generate protective immunity against several pathogens with a single dose Polyamines (polyethylene imine) condense RNA via inter-action with the phosphate backbone, resulting in compact nanoparticles that protect from nucleases. Amines have pKa = 5-7, this facilitates release of RNA into the cytoplasm. Chahal et al, PNAS, 2016

28 Fully synthetic Single-dose Adjuvant-free Nanoparticle vaccine platform. Vaccines can be formed with multiple antigen-expressing replicons. After a single immunization vaccines elicit vital T-cell and ab responses and fully protect against lethal exposures to several deadly pathogens

29 -DCs can be targeted using i.v. administered RNA-lipoplexes (RNA-LPX)The LPX protects RNA and mediates its efficient uptake and expression of the encoded antigen by DCs and macrophages in various lymphoid compartments RNA-LPX encoding different antigens induce strong effector and memory T-cell responses (also in three melanoma patients) RNA-LPX - a universally applicable vaccine class for systemic DCs targeting and induction of both adaptive as well as type-I-IFN-mediated innate immune mechanisms (for cancer immunotherapy) Kranz et al, Nature, 2016

30 Get the shot! DNA vaccine