RESUMEN
The dynamics of immunity to infection in infants remain obscure. Here, we used a multi-omics approach to perform a longitudinal analysis of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infants and young children by analyzing blood samples and weekly nasal swabs collected before, during, and after infection with Omicron and non-Omicron variants. Infection stimulated robust antibody titers that, unlike in adults, showed no sign of decay for up to 300 days. Infants mounted a robust mucosal immune response characterized by inflammatory cytokines, interferon (IFN) α, and T helper (Th) 17 and neutrophil markers (interleukin [IL]-17, IL-8, and CXCL1). The immune response in blood was characterized by upregulation of activation markers on innate cells, no inflammatory cytokines, but several chemokines and IFNα. The latter correlated with viral load and expression of interferon-stimulated genes (ISGs) in myeloid cells measured by single-cell multi-omics. Together, these data provide a snapshot of immunity to infection during the initial weeks and months of life.
Asunto(s)
COVID-19 , SARS-CoV-2 , Adulto , Niño , Lactante , Humanos , Preescolar , SARS-CoV-2/metabolismo , Multiómica , Citocinas/metabolismo , Interferón-alfa , Inmunidad MucosaRESUMEN
In the 40 years since their discovery, dendritic cells (DCs) have been recognized as central players in immune regulation. DCs sense microbial stimuli through pathogen-recognition receptors (PRRs) and decode, integrate, and present information derived from such stimuli to T cells, thus stimulating immune responses. DCs can also regulate the quality of immune responses. Several functionally specialized subsets of DCs exist, but DCs also display functional plasticity in response to diverse stimuli. In addition to sensing pathogens via PRRs, emerging evidence suggests that DCs can also sense stress signals, such as amino acid starvation, through ancient stress and nutrient sensing pathways, to stimulate adaptive immunity. Here, I discuss these exciting advances in the context of a historic perspective on the discovery of DCs and their role in immune regulation. I conclude with a discussion of emerging areas in DC biology in the systems immunology era and suggest that the impact of DCs on immunity can be usefully contextualized in a hierarchy-of-organization model in which DCs, their receptors and signaling networks, cell-cell interactions, tissue microenvironment, and the host macroenvironment represent different levels of the hierarchy. Immunity or tolerance can then be represented as a complex function of each of these hierarchies.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Animales , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Selección Clonal Mediada por Antígenos , Resistencia a la Enfermedad , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno/inmunología , Humanos , Linfocitos/citología , Linfocitos/inmunología , Linfocitos/metabolismo , Estrés FisiológicoRESUMEN
During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.
Asunto(s)
Anticuerpos Antivirales , Vacuna BNT162 , COVID-19 , Centro Germinal , Antígenos Virales , COVID-19/prevención & control , Humanos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus , VacunaciónRESUMEN
Bacille Calmette-Guérin (BCG) vaccination can confer nonspecific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. We show that mice vaccinated intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 B.1.351) or PR8 influenza. Protection was first evident between 14 and 21 d post-vaccination and lasted â¼3 months. Notably, BCG induced a biphasic innate response and robust antigen-specific type 1 helper T cell (TH1 cell) responses in the lungs. MyD88 signaling was essential for innate and TH1 cell responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or interferon (IFN)-γ activity before infection obliterated innate activation and protection. Single-cell and spatial transcriptomics revealed CD4-dependent expression of IFN-stimulated genes in lung myeloid and epithelial cells. Notably, BCG also induced protection against weight loss after mouse-adapted SARS-CoV-2 BA.5, SARS-CoV and SHC014 coronavirus infections. Thus, BCG elicits integrated organ immunity, where CD4+ T cells feed back on tissue myeloid and epithelial cells to imprint prolonged and broad innate antiviral resistance.
Asunto(s)
Inmunidad Adaptativa , Vacuna BCG , Animales , Ratones , Humanos , Retroalimentación , Vacunación , Pérdida de Peso , Antivirales , Inmunidad InnataRESUMEN
Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.
Asunto(s)
Epigenómica , Inmunidad/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Análisis de la Célula Individual , Transcripción Genética , Vacunación , Adolescente , Adulto , Antibacterianos/farmacología , Antígenos CD34/metabolismo , Antivirales/farmacología , Reprogramación Celular , Cromatina/metabolismo , Citocinas/biosíntesis , Combinación de Medicamentos , Femenino , Regulación de la Expresión Génica , Histonas/metabolismo , Humanos , Inmunidad Innata/genética , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Interferón Tipo I/metabolismo , Masculino , Células Mieloides/metabolismo , Polisorbatos/farmacología , Escualeno/farmacología , Receptores Toll-Like/metabolismo , Factor de Transcripción AP-1/metabolismo , Transcriptoma/genética , Adulto Joven , alfa-Tocoferol/farmacologíaRESUMEN
Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.
RESUMEN
Our previous study using systems vaccinology identified an association between the sterol regulatory binding protein (SREBP) pathway and humoral immune response to vaccination in humans. To investigate the role of SREBP signaling in modulating immune responses, we generated mice with B cell- or CD11c+ antigen-presenting cell (APC)-specific deletion of SCAP, an essential regulator of SREBP signaling. Ablation of SCAP in CD11c+ APCs had no effect on immune responses. In contrast, SREBP signaling in B cells was critical for antibody responses, as well as the generation of germinal centers,memory B cells and bone marrow plasma cells. SREBP signaling was required for metabolic reprogramming in activated B cells. Upon mitogen stimulation, SCAP-deficient B cells could not proliferate and had decreased lipid rafts. Deletion of SCAP in germinal center B cells using AID-Cre decreased lipid raft content and cell cycle progression. These studies provide mechanistic insights coupling sterol metabolism with the quality and longevity of humoral immunity.
Asunto(s)
Proteínas Portadoras , Linfoma de Células B , Esteroles , Animales , Humanos , Ratones , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Esteroles/metabolismo , Linfoma de Células B/metabolismoRESUMEN
Several studies have shown that the pre-vaccination immune state is associated with the antibody response to vaccination. However, the generalizability and mechanisms that underlie this association remain poorly defined. Here, we sought to identify a common pre-vaccination signature and mechanisms that could predict the immune response across 13 different vaccines. Analysis of blood transcriptional profiles across studies revealed three distinct pre-vaccination endotypes, characterized by the differential expression of genes associated with a pro-inflammatory response, cell proliferation, and metabolism alterations. Importantly, individuals whose pre-vaccination endotype was enriched in pro-inflammatory response genes known to be downstream of nuclear factor-kappa B showed significantly higher serum antibody responses 1 month after vaccination. This pro-inflammatory pre-vaccination endotype showed gene expression characteristic of the innate activation state triggered by Toll-like receptor ligands or adjuvants. These results demonstrate that wide variations in the transcriptional state of the immune system in humans can be a key determinant of responsiveness to vaccination.
Asunto(s)
Formación de Anticuerpos , Vacunas , Humanos , Vacunación , Adyuvantes Inmunológicos , Inmunidad InnataRESUMEN
Systems vaccinology has defined molecular signatures and mechanisms of immunity to vaccination. However, comparative analysis of immunity to different vaccines is lacking. We integrated transcriptional data of over 3,000 samples, from 820 adults across 28 studies of 13 vaccines and analyzed vaccination-induced signatures of antibody responses. Most vaccines induced signatures of innate immunity and plasmablasts at days 1 and 7, respectively, after vaccination. However, the yellow fever vaccine induced an early transient signature of T and B cell activation at day 1, followed by delayed antiviral/interferon and plasmablast signatures that peaked at days 7 and 14-21, respectively. Thus, there was no evidence for a 'universal signature' that predicted antibody response to all vaccines. However, accounting for the asynchronous nature of responses, we defined a time-adjusted signature that predicted antibody responses across vaccines. These results provide a transcriptional atlas of immunity to vaccination and define a common, time-adjusted signature of antibody responses.
Asunto(s)
Formación de Anticuerpos , Vacunas , Adulto , Humanos , Formación de Anticuerpos/genética , Perfilación de la Expresión Génica/métodos , Vacunación , Inmunidad Innata , Anticuerpos AntiviralesRESUMEN
Despite the success of the BNT162b2 mRNA vaccine, the immunological mechanisms that underlie its efficacy are poorly understood. Here we analyzed the innate and adaptive responses to BNT162b2 in mice, and show that immunization stimulated potent antibody and antigen-specific T cell responses, as well as strikingly enhanced innate responses after secondary immunization, which was concurrent with enhanced serum interferon (IFN)-γ levels 1 d following secondary immunization. Notably, we found that natural killer cells and CD8+ T cells in the draining lymph nodes are the major producers of this circulating IFN-γ. Analysis of knockout mice revealed that induction of antibody and T cell responses to BNT162b2 was not dependent on signaling via Toll-like receptors 2, 3, 4, 5 and 7 nor inflammasome activation, nor the necroptosis or pyroptosis cell death pathways. Rather, the CD8+ T cell response induced by BNT162b2 was dependent on type I interferon-dependent MDA5 signaling. These results provide insights into the molecular mechanisms by which the BNT162b2 vaccine stimulates immune responses.
Asunto(s)
Linfocitos T CD8-positivos , Vacunas , Inmunidad Adaptativa , Animales , Vacuna BNT162 , Humanos , Inmunidad Innata , Ratones , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.
Asunto(s)
Antibacterianos/farmacología , Anticuerpos Antivirales/inmunología , Microbioma Gastrointestinal/fisiología , Inmunidad/efectos de los fármacos , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Adolescente , Adulto , Formación de Anticuerpos , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Voluntarios Sanos , Humanos , Inmunogenicidad Vacunal/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Masculino , Adulto JovenRESUMEN
T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring 1 week post the second vaccination (boost), whereas CD8+ T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination.
Asunto(s)
COVID-19 , Vacunas , Humanos , Linfocitos T CD8-positivos , Vacuna BNT162 , SARS-CoV-2 , Vacunación , Anticuerpos AntiviralesRESUMEN
Herpes zoster (shingles) causes significant morbidity in immune compromised hosts and older adults. Whereas a vaccine is available for prevention of shingles, its efficacy declines with age. To help to understand the mechanisms driving vaccinal responses, we constructed a multiscale, multifactorial response network (MMRN) of immunity in healthy young and older adults immunized with the live attenuated shingles vaccine Zostavax. Vaccination induces robust antigen-specific antibody, plasmablasts, and CD4+ T cells yet limited CD8+ T cell and antiviral responses. The MMRN reveals striking associations between orthogonal datasets, such as transcriptomic and metabolomics signatures, cell populations, and cytokine levels, and identifies immune and metabolic correlates of vaccine immunity. Networks associated with inositol phosphate, glycerophospholipids, and sterol metabolism are tightly coupled with immunity. Critically, the sterol regulatory binding protein 1 and its targets are key integrators of antibody and T follicular cell responses. Our approach is broadly applicable to study human immunity and can help to identify predictors of efficacy as well as mechanisms controlling immunity to vaccination.
Asunto(s)
Vacuna contra el Herpes Zóster/inmunología , Inmunidad Adaptativa , Adulto , Anciano , Envejecimiento , Formación de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Fosfatos de Inositol/inmunología , Estudios Longitudinales , Masculino , Metabolómica , Persona de Mediana Edad , Caracteres Sexuales , Esteroles/metabolismo , Carga ViralRESUMEN
The 1st International Conference on Human &Translational Immunology convened in Rhodes, Greece, and provided a venue for stimulating scientific discussions on the human immune system.
Asunto(s)
Alergia e Inmunología , Investigación Biomédica Traslacional , Envejecimiento/inmunología , Enfermedades Autoinmunes/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Sistema Inmunológico/inmunología , Inmunidad Mucosa , Inmunogenicidad Vacunal , Inmunoterapia/métodos , Inmunoterapia/tendencias , Internacionalidad , Neoplasias/inmunología , Neoplasias/terapia , VacunaciónRESUMEN
The emergency use authorization of two mRNA vaccines in less than a year from the emergence of SARS-CoV-2 represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems vaccinology approach to comprehensively profile the innate and adaptive immune responses of 56 healthy volunteers who were vaccinated with the Pfizer-BioNTech mRNA vaccine (BNT162b2). Vaccination resulted in the robust production of neutralizing antibodies against the wild-type SARS-CoV-2 (derived from 2019-nCOV/USA_WA1/2020) and, to a lesser extent, the B.1.351 strain, as well as significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. Booster vaccination stimulated a notably enhanced innate immune response as compared to primary vaccination, evidenced by (1) a greater frequency of CD14+CD16+ inflammatory monocytes; (2) a higher concentration of plasma IFNγ; and (3) a transcriptional signature of innate antiviral immunity. Consistent with these observations, our single-cell transcriptomics analysis demonstrated an approximately 100-fold increase in the frequency of a myeloid cell cluster enriched in interferon-response transcription factors and reduced in AP-1 transcription factors, after secondary immunization. Finally, we identified distinct innate pathways associated with CD8 T cell and neutralizing antibody responses, and show that a monocyte-related signature correlates with the neutralizing antibody response against the B.1.351 variant. Collectively, these data provide insights into the immune responses induced by mRNA vaccination and demonstrate its capacity to prime the innate immune system to mount a more potent response after booster immunization.
Asunto(s)
Inmunidad Adaptativa , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Inmunidad Innata , Linfocitos T/inmunología , Vacunología , Adulto , Anciano , Anticuerpos Neutralizantes/inmunología , Autoanticuerpos/inmunología , Vacuna BNT162 , Vacunas contra la COVID-19/administración & dosificación , Femenino , Humanos , Inmunización Secundaria , Masculino , Persona de Mediana Edad , Análisis de la Célula Individual , Glicoproteína de la Espiga del Coronavirus/inmunología , Transcripción Genética , Transcriptoma/genética , Adulto JovenRESUMEN
Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m6A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific T cell activation, antibody production, and anti-tumor immunity in vivo, and m6A modification abrogates immune gene activation and adjuvant activity. m6A reader YTHDF2 sequesters m6A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m6A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.
Asunto(s)
Adenosina/análogos & derivados , Inmunidad Innata , Melanoma Experimental/terapia , ARN Circular/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina/administración & dosificación , Adenosina/inmunología , Adenosina/metabolismo , Adyuvantes Inmunológicos/administración & dosificación , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proteína 58 DEAD Box/inmunología , Proteína 58 DEAD Box/metabolismo , Femenino , Células HEK293 , Células HeLa , Humanos , Inmunización , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferones/inmunología , Interferones/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones Endogámicos C57BL , Poliubiquitina/inmunología , Poliubiquitina/metabolismo , Multimerización de Proteína , ARN Circular/administración & dosificación , ARN Circular/metabolismo , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/metabolismo , Receptores Inmunológicos , UbiquitinaciónRESUMEN
The unfolded protein response (UPR) has traditionally been viewed as an adaptive response triggered by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and aimed at restoring ER function. The UPR can also be an anticipatory response that is activated well before the disruption of protein homeostasis. UPR signaling intersects at many levels with the innate and adaptive immune responses. In some types of cells of the immune system, such as dendritic cells (DCs) and B cells, particular sensors that detect the UPR seem to be constitutively active in the absence of induction of the traditional UPR gene program and are necessary for antigen presentation and immunoglobulin synthesis. The UPR also influences signaling via Toll-like receptors (TLRs) and activation of the transcription factor NF-κB, and some pathogens subvert the UPR. This Review summarizes these emerging noncanonical functions of the UPR in immunity.
Asunto(s)
Inmunidad Adaptativa/inmunología , Inmunidad Innata/inmunología , Transducción de Señal/inmunología , Respuesta de Proteína Desplegada/inmunología , Animales , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/metabolismo , Modelos Inmunológicos , FN-kappa B/inmunología , FN-kappa B/metabolismo , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
Many vaccines induce protective immunity via antibodies. Systems biology approaches have been used to determine signatures that can be used to predict vaccine-induced immunity in humans, but whether there is a 'universal signature' that can be used to predict antibody responses to any vaccine is unknown. Here we did systems analyses of immune responses to the polysaccharide and conjugate vaccines against meningococcus in healthy adults, in the broader context of published studies of vaccines against yellow fever virus and influenza virus. To achieve this, we did a large-scale network integration of publicly available human blood transcriptomes and systems-scale databases in specific biological contexts and deduced a set of transcription modules in blood. Those modules revealed distinct transcriptional signatures of antibody responses to different classes of vaccines, which provided key insights into primary viral, protein recall and anti-polysaccharide responses. Our results elucidate the early transcriptional programs that orchestrate vaccine immunity in humans and demonstrate the power of integrative network modeling.