Virus-host interactome: Putting the accent on how it changes

[EN] Viral infections are extremely complex processes that could only be well understood by precisely characterizing the interaction networks between the virus and the host components. In recent years, much effort has gone in this directionwith the aimof unveiling themolecular basis of viral pathology. These networks are mostly formed by viral and host proteins, and are expected to be dynamic bothwith time and space (i.e., with the progression of infection, as well as with the virus and host genotypes; what we call plastodynamic). This largely overlooked spatio-temporal evolution urgently calls for a change both in the conceptual paradigms and experimental techniques used so far to characterize virus-host interactions. More generally, molecular plasticity and temporal dynamics are unavoidable components of themechanisms that underlie any complex disease; components whose understandingwill eventually enhance our ability to modulate those networkswith the aimof improving disease treatments.This work is supported by the grants BFU2015-66894-P (to G.R.), BI02014-54269-R (to J-A.D.) and BFU2015-65037-P (to S.F.E.) from the Ministerio de Economia, Industria y Competitividad, and by the grant PROMETEOII/2014/021 from the Generalitat Valenciana (to S.F.E. and J-A.D.).Rodrigo Tarrega, G.; Daros Arnau, JA.; Elena Fito, SF. (2017). Virus-host interactome: Putting the accent on how it changes. Journal of Proteomics. 156:1-4. https://doi.org/10.1016/j.jprot.2016.12.007S1415

Temporal Dynamics of Host Molecular Responses Differentiate Symptomatic and Asymptomatic Influenza A Infection

Exposure to influenza viruses is necessary, but not sufficient, for healthy human hosts to develop symptomatic illness. The host response is an important determinant of disease progression. In order to delineate host molecular responses that differentiate symptomatic and asymptomatic Influenza A infection, we inoculated 17 healthy adults with live influenza (H3N2/Wisconsin) and examined changes in host peripheral blood gene expression at 16 timepoints over 132 hours. Here we present distinct transcriptional dynamics of host responses unique to asymptomatic and symptomatic infections. We show that symptomatic hosts invoke, simultaneously, multiple pattern recognition receptors-mediated antiviral and inflammatory responses that may relate to virus-induced oxidative stress. In contrast, asymptomatic subjects tightly regulate these responses and exhibit elevated expression of genes that function in antioxidant responses and cell-mediated responses. We reveal an ab initio molecular signature that strongly correlates to symptomatic clinical disease and biomarkers whose expression patterns best discriminate early from late phases of infection. Our results establish a temporal pattern of host molecular responses that differentiates symptomatic from asymptomatic infections and reveals an asymptomatic host-unique non-passive response signature, suggesting novel putative molecular targets for both prognostic assessment and ameliorative therapeutic intervention in seasonal and pandemic influenza

Plasticity in gene expression programmes of dendritic cells responding to antigens.

Dendritic cells (DCs) are professional antigen presenting cells whose function is to initiate and shape an appropriate adaptive immune response. This requires an ability to distinguish differences between whole pathogens, in order to orchestrate effective downstream immunological outcomes. However, cellular re-programming of DC functions during these events are not well understood. A paradigm of dendritic cell biology is that DCs have two modes of function that relate to their differentiation states. An immature DC functions as an immune sentinel, to monitor and interrogate its surroundings for pathogens. Encounter with such stimuli results in a process termed "maturation", where DCs acquire the properties of effective antigen presenting cells. However, this process of differentiation is complex. In this thesis, gene expression profiling of DCs exposed to pathogen components has revealed three distinct phases of maturation, with statistically significant expression of subsets of genes characterising these phases. Transcriptional regulation of the signalling pathways involving p38 and ERK MAP kinases important to DC function were identified. Specific inhibitors of p38 and ERK confirmed their differential role in DC maturation, with p38 activity being necessary for the initiation of DC maturation, whilst ERK activity persists to maintain DC survival. Concurrent with the core maturation process is the DCs' ability to differentially respond to pathogens. Gene expression analysis of DCs exposed to whole viruses supports the model of DC plasticity to different pathogenic stimuli. Using exploratory cluster analysis and a novel vector algebra method, core and pathogen-specific gene expression programmes were identified. The programmes involving the differential regulation of cytokines were confirmed at the transcript level and at the protein level. Together these data show that DCs mature to effective antigen presenting cells via an orchestrated pattern of at least three gene expression programmes. Superimposed on this core maturation response are pathogen-specific transcriptional programmes. Therefore, we conclude that DCs can translate different pathogenic stimuli into core DC maturation and pathogen-specific responses that together shape an appropriate adaptive immune response

Selective transmission of R5 HIV-1 variants: where is the gatekeeper?

To enter target cells HIV-1 uses CD4 and a coreceptor. In vivo the coreceptor function is provided either by CCR5 (for R5) or CXCR4 (for X4 HIV-1). Although both R5 and X4 HIV-1 variants are present in body fluids (semen, blood, cervicovaginal and rectal secretions), R5 HIV-1 appears to transmit infection and dominates early stages of HIV disease. Moreover, recent sequence analysis of virus in acute infection shows that, in the majority of cases of transmission, infection is initiated by a single virus. Therefore, the existence of a “gatekeeper” that selects R5 over X4 HIV-1 and that operates among R5 HIV-1 variants has been suggested. In the present review we consider various routes of HIV-transmission and discuss potential gatekeeping mechanisms associated with each of these routes. Although many mechanisms have been identified none of them explains the almost perfect selection of R5 over X4 in HIV-1 transmission. We suggest that instead of one strong gatekeeper there are multiple functional gatekeepers and that their superimposition is sufficient to protect against X4 HIV-1 infection and potentially select among R5 HIV-1 variants. In conclusion, we propose that the principle of multiple barriers is more general and not restricted to protection against X4 HIV-1 but rather can be applied to other phenomena when one factor has a selective advantage over the other(s). In the case of gatekeepers for HIV-1 transmission, the task is to identify them and to decipher their molecular mechanisms. Knowledge of the gatekeepers‘ localization and function may enable us to enhance existing barriers against R5 transmission and to erect the new ones against all HIV-1 variants

Cellular immune ontogeny and birth transcriptome in HIV-exposed uninfected infants

Background. In some regions of Sub-Saharan Africa, up to 30% of newborns are born to mothers infected with human immunodeficiency virus (HIV). Maternal antiretroviral treatment (ART) has reduced vertical transmission to lower than 1%. Despite the success of prevention of mother-tochild transmission (PMTCT) programmes, a large number of children born to these mothers are exposed to HIV and antiretrovirals (ARVs) in utero yet remain uninfected. These individuals, known as children who are HIV-exposed and uninfected (cHEU), succumb to higher rates of disease morbidity compared to children who are HIV-unexposed (cHU) which suggests altered immunity in the cHEU. Differences in the numbers and function of cells of the innate and adaptive immune system have been documented in cHEU—though not consistently. While vaccine-induced antibody responses are robust in cHEU, data on potential cell mediated perturbations to vaccine antigens remains conflicting. This is in part due to inherent inter-cohort variation and differences in ART therapy strategies, feeding practices between cohorts and the assays used measure cellmediated responses. We leveraged two independent cohorts from Nigeria and South Africa of mother-infant pairs receiving antenatal and postnatal care all under Option B+ PMTCT. All HEU infants received pre-exposure prophylaxis for 6 weeks and the majority were exclusively breastfed until 6 months of age. We applied the same assays in both cohorts to test the hypotheses that HEU have altered T cell immunity compared to HU controls and distinct transcriptomic signatures at birth. These were tested in three distinct aims: 1) To identify transcriptional signatures at baseline that delineate cHEU from cHU 2) To compare the expression of surface marker broadly defining activated or regulatory phenotypes and the expression of intracellular markers of T cell function between cHEU and cHU over the first 9 months of life. 3) To characterise how differences in the immunising strains of Bacille Calmette-Gu'erin (BCG), the first vaccine received in these infants, impacts T cell immunity to both mycobacterial and non-mycobacterial antigens in cHEU and cHU. Methods. Two birth cohorts from Jos, Nigeria and Cape Town (CT), South Africa were recruited into this study as part of a larger parent study that aims to identify biological determinants of protection from mother-to-child transmission of HIV (Innate, Adaptive and Mucosal Immune Responses in Infants/INFANT study: HREC 285/2012). Infant blood was collected at several time points from birth to 36 weeks of life for immunological assays. Whole blood, collected at birth was preserved in PAXgene fluid for downstream messenger ribonucleic acid (mRNA) transcript analyses. Other whole blood samples were fixed and cryopreserved either directly ex vivo or after re-stimulation within 1 hour of phlebotomy with BCG, Tetanus Toxoid (TT), Bordetella pertussis (BP) antigens and Phytohemagglutinin (PHA). Multi-parameter flow cytometry was used to measure batched whole blood samples for (i) markers of T cell regulation and activation directly ex vivo, markers of T cell gut homing and proliferation—a proxy for HIV susceptibility, and (ii) vaccine-induced Th1 cytokine expression (IFN-, TNF-a, IL-2) and memory maturation. Cytokine responses were profiled for polyfunctionality by SPICE analysis and complemented by the COMPASS algorithm. Transcriptional profiling of whole blood at birth was done by RNA sequencing and differentially expressed genes were reported for absolute fold change of normalized counts were < 1.5 with FDR set at 0.05 using the DESeq2 package in R. Gene-set enrichment analysis (GSEA) was used to identify enriched or repressed gene pathways for absolute normalised effect sizes < 1.5 with FDR set at 0.05. Longitudinal analyses used a mixed effects ANOVA with time and HIV exposure as explanatory variables. Cross-sectional analyses comparing HIV exposure groups used Wilcoxon Ranked Sum Test, with p< 0.05 considered significant after multiple correction adjustment by Holm's step-down method. Results. Aim 1: A small set of DEGs were found between HEUs and HU groups at birth, 3 of which were upregulated and 12 that were downregulated. Among the upregulated genes, two are homologues of the arrestins: ARRDC4 (2.3 fold, adjusted p-adj< 0.001) and TXNIP (1.4 fold, padj< 0.001). Gene-set enrichment analysis however, showed no significant enrichment or suppression of gene pathways in HEUs. Aim 2: HIV/ARV exposure did not have an interaction effect with age (all time points) in explaining the frequencies of T cell markers ex vivo in a mixed-effects model. In cross-sectional unadjusted analyses however, trends towards increased median frequencies of markers of activation in the HEU group compared to HU controls were observed for specific ages: at birth (%CD8+HLA-DR+: 0.12 vs. 0.01, p=0.05), at week 7 (%CD8+CD25+: 0.13 vs. 0.04, p=0.01 and %CD8+HLA-DR+: 0.84 vs. 0.07, p=0.01) and at week 36 (%CD8+CD25+: 0.52 vs. 0.03, p< 0.001 and %CD8+HLA-DR+: 0.81 vs. 0.17, p=0.003). When adjusting for multiple comparisons, only CD25 expression remained significant on CD8+ T cells at week 36 (p-adj =0.04). The magnitudes of cytokine responses by T cells to vaccine antigens did not differ between HEU and HU infants however, transient differences in the polyfunctional profile of cells was observed at week 1 for mycobacterial-specific Th1 profiles in CT infants (p=0.002) by SPICE analysis. There were later differences at week 7 for BP-specific Th1 profiles in Jos infants (p=0.01) and at week 36 for BP-specific Th1 profiles in CT infants (p=0.03). The more robust COMPASS algorithm only detected a trend towards increased polyfunctional scores to BP responses in CT infants at week 36 (p=0.03). Aim 3: BCG immunising strain impacted the magnitudes and quality of responses to mycobacterial and non-mycobacterial vaccine antigens irrespective of HIV exposure status. Most significantly, at week 7, BCG-Denmark induced higher mycobacterial-specific frequencies of CD4 Th1 cytokines compared to Bulgarian (p< 0.001) and Russian strains (and (p< 0.001). BCGDenmark induced greater triple cytokine profiles to mycobacterial antigen compared to Bulgarian (p< 0.001) and Russian (p< 0.001) strains in SPICE analyses and the resulted were confirmed by COMPASS algorithm polyfunctional scores. Furthermore, BCG-Denmark significantly enhanced antigenicity to TT and BP vaccines. Conclusion. Transient differences exist in the frequencies of CD25 expressing CD8 T cells between HEU and HU groups, however other readouts of immunity suggest that in the context of effective PMTCT and exclusive breastfeeding practices, HEU infants are indistinguishable from their HIV unexposed peers