Molecular Signature of Monocytes Shaped by the Shigella sonnei 1790-Generalized Modules for Membrane Antigens Vaccine.

Shigellosis, an acute gastroenteritis infection caused by Shigella species, remains a public health burden in developing countries. Recently, many outbreaks due to Shigella sonnei multidrug-resistant strains have been reported in high-income countries, and the lack of an effective vaccine represents a major hurdle to counteract this bacterial pathogen. Vaccine candidates against Shigella sonnei are under clinical development, including a Generalized Modules for Membrane Antigens (GMMA)-based vaccine. The mechanisms by which GMMA-based vaccines interact and activate human immune cells remain elusive. Our previous study provided the first evidence that both adaptive and innate immune cells are targeted and functionally shaped by the GMMA-based vaccine. Here, flow cytometry and confocal microscopy analysis allowed us to identify monocytes as the main target population interacting with the S. sonnei 1790-GMMA vaccine on human peripheral blood. In addition, transcriptomic analysis of this cell population revealed a molecular signature induced by 1790-GMMA mostly correlated with the inflammatory response and cytokine-induced processes. This also impacts the expression of genes associated with macrophages' differentiation and T cell regulation, suggesting a dual function for this vaccine platform both as an antigen carrier and as a regulator of immune cell activation and differentiation.

Systems analysis of human responses to an aluminium hydroxide-adsorbed TLR7 agonist (AS37) adjuvanted vaccine reveals a dose-dependent and specific activation of the interferon-mediated antiviral response.

The candidate Adjuvant System AS37 contains a synthetic toll-like receptor agonist (TLR7a) adsorbed to alum. In a phase I study (NCT02639351), healthy adults were randomised to receive one dose of licensed alum-adjuvanted meningococcal serogroup C (MenC-CRM197) conjugate vaccine (control) or MenC-CRM197 conjugate vaccine adjuvanted with AS37 (TLR7a dose 12.5, 25, 50 or 100 µg). A subset of 66 participants consented to characterisation of peripheral whole blood transcriptomic responses, systemic cytokine/chemokine responses and multiple myeloid and lymphoid cell responses as exploratory study endpoints. Blood samples were collected pre-vaccination, 6 and 24 h post-vaccination, and 3, 7, 28 and 180 days post-vaccination. The gene expression profile in whole blood showed an early, AS37-specific transcriptome response that peaked at 24 h, increased with TLR7a dose up to 50 µg and generally resolved within one week. Five clusters of differentially expressed genes were identified, including those involved in the interferon-mediated antiviral response. Evaluation of 30 cytokines/chemokines by multiplex assay showed an increased level of interferon-induced chemokine CXCL10 (IP-10) at 24 h and 3 days post-vaccination in the AS37-adjuvanted vaccine groups. Increases in activated plasmacytoid dendritic cells (pDC) and intermediate monocytes were detected 3 days post-vaccination in the AS37-adjuvanted vaccine groups. T follicular helper (Tfh) cells increased 7 days post-vaccination and were maintained at 28 days post-vaccination, particularly in the AS37-adjuvanted vaccine groups. Moreover, most of the subjects that received vaccine containing 25, 50 and 100 µg TLR7a showed an increased MenC-specific memory B cell responses versus baseline. These data show that the adsorption of TLR7a to alum promotes an immune signature consistent with TLR7 engagement, with up-regulation of interferon-inducible genes, cytokines and frequency of activated pDC, intermediate monocytes, MenC-specific memory B cells and Tfh cells. TLR7a 25-50 µg can be considered the optimal dose for AS37, particularly for the adjuvanted MenC-CRM197 conjugate vaccine.

Single-Cell Analysis of Antigen-Specific CD8+ T-Cell Transcripts Reveals Profiles Specific to mRNA or Adjuvanted Protein Vaccines.

CD8+ T cells play a key role in mediating protective immunity after immune challenges such as infection or vaccination. Several subsets of differentiated CD8+ T cells have been identified, however, a deeper understanding of the molecular mechanism that underlies T-cell differentiation is lacking. Conventional approaches to the study of immune responses are typically limited to the analysis of bulk groups of cells that mask the cells' heterogeneity (RNA-seq, microarray) and to the assessment of a relatively limited number of biomarkers that can be evaluated simultaneously at the population level (flow and mass cytometry). Single-cell analysis, on the other hand, represents a possible alternative that enables a deeper characterization of the underlying cellular heterogeneity. In this study, a murine model was used to characterize immunodominant hemagglutinin (HA533-541)-specific CD8+ T-cell responses to nucleic- and protein-based influenza vaccine candidates, using single-cell sorting followed by transcriptomic analysis. Investigation of single-cell gene expression profiles enabled the discovery of unique subsets of CD8+ T cells that co-expressed cytotoxic genes after vaccination. Moreover, this method enabled the characterization of antigen specific CD8+ T cells that were previously undetected. Single-cell transcriptome profiling has the potential to allow for qualitative discrimination of cells, which could lead to novel insights on biological pathways involved in cellular responses. This approach could be further validated and allow for more informed decision making in preclinical and clinical settings.

Systematic characterization of human response to H1N1 influenza vaccination through the construction and integration of personalized transcriptome response profiles.

Gene expression data is commonly used in vaccine studies to characterize differences between treatment groups or sampling time points. Group-wise comparisons of the transcriptional perturbations induced by vaccination have been applied extensively for investigating the mechanisms of action of vaccines. Such approaches, however, may not be sensitive enough for detecting changes occurring within a minority of the population under investigation or in single individuals. In this study, we developed a data analysis framework to characterize individual subject response profiles in the context of repeated measure experiments, which are typical of vaccine mode of action studies. Following the definition of the methodology, this was applied to the analysis of human transcriptome responses induced by vaccination with a subunit influenza vaccine. Results highlighted a substantial heterogeneity in how different subjects respond to vaccination. Moreover, the extent of transcriptional modulation experienced by each individual subject was found to be associated with the magnitude of vaccine-specific functional antibody response, pointing to a mechanistic link between genes involved in protein production and innate antiviral response. Overall, we propose that the improved characterization of the intersubject heterogeneity, enabled by our approach, can help driving the improvement and optimization of current and next-generation vaccines.

Literature Mining and Mechanistic Graphical Modelling to Improve mRNA Vaccine Platforms.

RNA vaccines represent a milestone in the history of vaccinology. They provide several advantages over more traditional approaches to vaccine development, showing strong immunogenicity and an overall favorable safety profile. While preclinical testing has provided some key insights on how RNA vaccines interact with the innate immune system, their mechanism of action appears to be fragmented amid the literature, making it difficult to formulate new hypotheses to be tested in clinical settings and ultimately improve this technology platform. Here, we propose a systems biology approach, based on the combination of literature mining and mechanistic graphical modeling, to consolidate existing knowledge around mRNA vaccines mode of action and enhance the translatability of preclinical hypotheses into clinical evidence. A Natural Language Processing (NLP) pipeline for automated knowledge extraction retrieved key biological evidences that were joined into an interactive mechanistic graphical model representing the chain of immune events induced by mRNA vaccines administration. The achieved mechanistic graphical model will help the design of future experiments, foster the generation of new hypotheses and set the basis for the development of mathematical models capable of simulating and predicting the immune response to mRNA vaccines.

Transcriptional profiles of adjuvanted hepatitis B vaccines display variable interindividual homogeneity but a shared core signature.

The current routine use of adjuvants in human vaccines provides a strong incentive to increase our understanding of how adjuvants differ in their ability to stimulate innate immunity and consequently enhance vaccine immunogenicity. Here, we evaluated gene expression profiles in cells from whole blood elicited in naive subjects receiving the hepatitis B surface antigen formulated with different adjuvants. We identified a core innate gene signature emerging 1 day after the second vaccination and that was shared by the recipients of vaccines formulated with adjuvant systems AS01B, AS01E, or AS03. This core signature associated with the magnitude of the hepatitis B surface-specific antibody response and was characterized by positive regulation of genes associated with interferon-related responses or the innate cell compartment and by negative regulation of natural killer cell-associated genes. Analysis at the individual subject level revealed that the higher immunogenicity of AS01B-adjuvanted vaccine was linked to its ability to induce this signature in most vaccinees even after the first vaccination. Therefore, our data suggest that adjuvanticity is not strictly defined by the nature of the receptors or signaling pathways it activates but by the ability of the adjuvant to consistently induce a core inflammatory signature across individuals.

Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

Adjuvant effect of TLR7 agonist adsorbed on aluminum hydroxide (AS37): A phase I randomized, dose escalation study of an AS37-adjuvanted meningococcal C conjugated vaccine.

An adjuvant system (AS37) has been developed containing a synthetic toll-like receptor agonist (TLR7a). We conducted a phase I randomized, observer-blind, dose-escalation study to assess the safety and immunogenicity of an investigational AS37-adjuvanted meningococcus C (MenC) conjugate vaccine in healthy adults (NCT02639351). A control group received a licensed MenC conjugate alum-adjuvanted vaccine. Eighty participants were randomized to receive one dose of control or investigational vaccine containing AS37 (TLR7a dose 12.5, 25, 50, 100 µg). All vaccines were well tolerated, apart from in the TLR7a 100 µg dose group, which had three reports (18.8%) of severe systemic adverse events. Four weeks after vaccination, human complement serum bactericidal assay seroresponse rates against MenC were 56-81% in all groups, and ELISA seroresponses were ≥81% for all AS37-adjuvanted vaccine groups (100% in 50 and 100 µg dose groups) and 88% in the control group. Antibody responses were maintained at six months after vaccination.

Will Systems Biology Deliver Its Promise and Contribute to the Development of New or Improved Vaccines? Systems Biology Views of Vaccine Innate and Adaptive Immunity.

During the last decade, several high-throughput technologies have been applied to gather deeper understanding on the biological events elicited by vaccination. The main goal of systems biology is to integrate different sources of data and extract biologically meaningful information. This holistic approach has provided new insights on the impact that the innate immune status has on vaccine responsiveness. Other factors like chronic infections, age, microbiome, and metabolism can influence the outcome of vaccination, and systems biology offers unique opportunities to expand our understanding of their role on the immune response. However, a few challenges that still need to be overcome will be discussed.

Innate transcriptional effects by adjuvants on the magnitude, quality, and durability of HIV envelope responses in NHPs.

Adjuvants have a critical role for improving vaccine efficacy against many pathogens, including HIV. Here, using transcriptional RNA profiling and systems serology, we assessed how distinct innate pathways altered HIV-specific antibody responses in nonhuman primates (NHPs) using 8 clinically based adjuvants. NHPs were immunized with a glycoprotein 140 HIV envelope protein (Env) and insoluble aluminum salts (alum), MF59, or adjuvant nanoemulsion (ANE) coformulated with or without Toll-like receptor 4 (TLR4) and 7 agonists. These were compared with Env administered with polyinosinic-polycytidylic acid:poly-L-lysine, carboxymethylcellulose (pIC:LC) or immune-stimulating complexes. Addition of the TLR4 agonist to alum enhanced upregulation of a set of inflammatory genes, whereas the TLR7 agonist suppressed expression of alum-responsive inflammatory genes and enhanced upregulation of antiviral and interferon (IFN) genes. Moreover, coformulation of the TLR4 or 7 agonists with alum boosted Env-binding titers approximately threefold to 10-fold compared with alum alone, but remarkably did not alter gene expression or enhance antibody titers when formulated with ANE. The hierarchy of adjuvant potency was established after the second of 4 immunizations. In terms of antibody durability, antibody titers decreased ∼10-fold after the final immunization and then remained stable after 65 weeks for all adjuvants. Last, Env-specific Fc-domain glycan structures and a series of antibody effector functions were assessed by systems serology. Antiviral/IFN gene signatures correlated with Fc-receptor binding across all adjuvant groups. This study defines the potency and durability of 8 different clinically based adjuvants in NHPs and shows how specific innate pathways can alter qualitative aspects of Env antibody function.

In-silico prediction and deep-DNA sequencing validation indicate phase variation in 115 Neisseria meningitidis genes.

BACKGROUND: The Neisseria meningitidis (Nm) chromosome shows a high abundance of simple sequence DNA repeats (SSRs) that undergo stochastic, reversible mutations at high frequency. This mechanism is reflected in an extensive phenotypic diversity that facilitates Nm adaptation to dynamic environmental changes. To date, phase-variable phenotypes mediated by SSRs variation have been experimentally confirmed for 26 Nm genes. RESULTS: Here we present a population-scale comparative genomic analysis that identified 277 genes and classified them into 52 strong, 60 moderate and 165 weak candidates for phase variation. Deep-coverage DNA sequencing of single colonies grown overnight under non-selective conditions confirmed the presence of high-frequency, stochastic variation in 115 of them, providing circumstantial evidence for their phase variability. We confirmed previous observations of a predominance of variable SSRs within genes for components located on the cell surface or DNA metabolism. However, in addition we identified an unexpectedly broad spectrum of other metabolic functions, and most of the variable SSRs were predicted to induce phenotypic changes by modulating gene expression at a transcriptional level or by producing different protein isoforms rather than mediating on/off translational switching through frameshifts. Investigation of the evolutionary history of SSR contingency loci revealed that these loci were inherited from a Nm ancestor, evolved independently within Nm, or were acquired by Nm through lateral DNA exchange. CONCLUSIONS: Overall, our results have identified a broader and qualitatively different phenotypic diversification of SSRs-mediated stochastic variation than previously documented, including its impact on central Nm metabolism.

Early Rise of Blood T Follicular Helper Cell Subsets and Baseline Immunity as Predictors of Persisting Late Functional Antibody Responses to Vaccination in Humans.

CD4+ T follicular helper cells (T(FH)) have been identified as the T-cell subset specialized in providing help to B cells for optimal activation and production of high affinity antibody. We recently demonstrated that the expansion of peripheral blood influenza-specific CD4(+)IL-21(+)ICOS1(+) T helper (T(H)) cells, three weeks after vaccination, associated with and predicted the rise of protective neutralizing antibodies to avian H5N1. In this study, healthy adults were vaccinated with plain seasonal trivalent inactivated influenza vaccine (TIIV), MF59(®)-adjuvanted TIIV (ATIIV), or saline placebo. Frequencies of circulating CD4(+) T(FH)1 ICOS(+) T(FH) cells and H1N1-specific CD4(+-)IL-21(+)ICOS(+) CXCR5(+) T(FH) and CXCR5(-) T(H) cell subsets were determined at various time points after vaccination and were then correlated with hemagglutination inhibition (HI) titers. All three CD4(+) T cell subsets expanded in response to TIIV and ATIIV, and peaked 7 days after vaccination. To demonstrate that these T(FH) cell subsets correlated with functional antibody titers, we defined an alternative endpoint metric, decorrelated HI (DHI), which removed any correlation between day 28/day 168 and day 0 HI titers, to control for the effect of preexisting immunity to influenza vaccine strains. The numbers of total circulating CD4(+)T(FH)1 ICOS(+) cells and of H1N1-specific CD4(+)IL-21(+)ICOS(+) CXCR5(+), measured at day 7, were significantly associated with day 28, and day 28 and 168 DHI titers, respectively. Altogether, our results show that CD4(+) T(FH) subsets may represent valuable biomarkers of vaccine-induced long-term functional immunity.

Global transcriptome analysis reveals small RNAs affecting Neisseria meningitidis bacteremia.

Most bacterial small RNAs (sRNAs) are post-transcriptional regulators involved in adaptive responses, controlling gene expression by modulating translation or stability of their target mRNAs often in concert with the RNA chaperone Hfq. Neisseria meningitides, the leading cause of bacterial meningitis, is able to adapt to different host niches during human infection. However, only a few sRNAs and their functions have been fully described to date. Recently, transcriptional expression profiling of N. meningitides in human blood ex vivo revealed 91 differentially expressed putative sRNAs. Here we expanded this analysis by performing a global transcriptome study after exposure of N. meningitides to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation). and we identified putative sRNAs that were differentially expressed in vitro. A set of 98 putative sRNAs was obtained by analyzing transcriptome data and 8 new sRNAs were validated, both by Northern blot and by primer extension techniques. Deletion of selected sRNAs caused attenuation of N. meningitides infection in the in vivo infant rat model, leading to the identification of the first sRNAs influencing meningococcal bacteremia. Further analysis indicated that one of the sRNAs affecting bacteremia responded to carbon source availability through repression by a GntR-like transcriptional regulator. Both the sRNA and the GntR-like regulator are implicated in the control of gene expression from a common network involved in energy metabolism.

Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination.

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis (Nm), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm. Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.

Building an insurance against modern pandemics.

The response to the first influenza pandemic of the 21st century benefited from the extensive preparation for an avian influenza pandemic and the mild nature of the 2009 A/H1N1 swine influenza virus. However, the pandemic demonstrated the limited ability to predict influenza pandemics, to anticipate levels of cross-protection, and to deliver vaccines in a timely manner, particularly to low income countries. The lessons learned from the 2009 H1N1 pandemic are of paramount importance to develop more effective preparations against future pandemics.