Analysis and Prediction of Pathogen Nucleic Acid Specificity for Toll-like Receptors in Vertebrates.

Identification of key sequence, expression and function related features of nucleic acid-sensing host proteins is of fundamental importance to understand the dynamics of pathogen-specific host responses. To meet this objective, we considered toll-like receptors (TLRs), a representative class of membrane-bound sensor proteins, from 17 vertebrate species covering mammals, birds, reptiles, amphibians, and fishes in this comparative study. We identified the molecular signatures of host TLRs that are responsible for sensing pathogen nucleic acids or other pathogen-associated molecular patterns (PAMPs), and potentially play important roles in host defence mechanism. Interestingly, our findings reveal that such host-specific features are directly related to the strand (single or double) specificity of nucleic acid from pathogens. However, during host-pathogen interactions, such features were unable to explain the pathogenic PAMP (i.e., DNA, RNA or other) selectivity, suggesting a more complex mechanism. Using these features, we developed a number of machine learning models, of which Random Forest achieved a high performance (94.57% accuracy) to predict strand specificity of TLRs from protein-derived features. We applied the trained model to propose strand specificity of some previously uncharacterized distinct fish-specific novel TLRs (TLR18, TLR23, TLR24, TLR25, TLR27).

A Long-Running Arms Race between APOBEC1 Genes and Retroviruses in Tetrapods.

Activation-induced cytidine deaminase/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) proteins are cytosine deaminases implicated in diverse biological functions. APOBEC1 (A1) proteins have long been thought to regulate lipid metabolism, whereas the evolutionary significance of A1 proteins in antiviral defense remains largely obscure. Endogenous retroviruses (ERVs) document past retroviral infections and are ubiquitous within the vertebrate genomes. Here, we identify the A1 gene repertoire, characterize the A1-mediated mutation footprints in ERVs, and interrogate the evolutionary arms race between A1 genes and ERVs across vertebrate species. We find that A1 genes are widely present in tetrapods, recurrently amplified and lost in certain lineages, suggesting that A1 genes might have originated during the early evolution of tetrapods. A1-mediated mutation footprints can be detected in ERVs across tetrapods. Moreover, A1 genes appear to have experienced episodic positive selection in many tetrapod lineages. Taken together, we propose that a long-running arms race between A1 genes and retroviruses might have persisted throughout the evolutionary course of tetrapods. IMPORTANCE APOBEC3 (A3) genes have been thought to function in defense against retroviruses, whereas the evolutionary significance of A1 proteins in antiviral defense remains largely obscure. In this study, we identify the A1 gene repertoire, characterize the A1-mediated mutation footprints in endogenous retroviruses (ERVs), and explore the evolutionary arms race between A1 genes and ERVs across vertebrate species. We found A1 proteins originated during the early evolution of tetrapods, and detected the footprints of A1-induced hypermutations in retroviral fossils. A1 genes appear to have experienced pervasive positive selection in tetrapods. Our study indicates a long-running arms race between A1 genes and retroviruses taking place throughout the evolutionary course of tetrapods.

Immunological Diversity Is a Cornerstone of Organismal Defense and Allorecognition across Metazoa.

The ongoing arms race between hosts and microbes has fueled the evolution of novel strategies for diversifying the molecules involved in immune responses. Characterization of immune systems from an ever-broadening phylogenetic range of organisms reveals that there are many mechanisms by which this diversity can be generated and maintained. Diversification strategies operate at the level of populations, genomes, genes, and even individual transcripts. Lineage-specific innovations have been cataloged within the immune systems of both invertebrates and vertebrates. Furthermore, somatic diversification of immune receptor genes has now been described in jawless vertebrates and some invertebrate species. In addition to pathogen detection, immunological diversity plays important roles in several distinct allorecognition systems. In this Brief Review, we highlight some of the evolutionary innovations employed by a variety of metazoan species to generate the molecular diversity required to detect a vast array of molecules in the context of both immune response and self/nonself-recognition.

IMGT® databases, related tools and web resources through three main axes of research and development.

IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org/, is at the forefront of the immunogenetics and immunoinformatics fields with more than 30 years of experience. IMGT® makes available databases and tools to the scientific community pertaining to the adaptive immune response, based on the IMGT-ONTOLOGY. We focus on the recent features of the IMGT® databases, tools, reference directories and web resources, within the three main axes of IMGT® research and development. Axis I consists in understanding the adaptive immune response, by deciphering the identification and characterization of the immunoglobulin (IG) and T cell receptor (TR) genes in jawed vertebrates. It is the starting point of the two other axes, namely the analysis and exploration of the expressed IG and TR repertoires based on comparison with IMGT reference directories in normal and pathological situations (Axis II) and the analysis of amino acid changes and functions of 2D and 3D structures of antibody and TR engineering (Axis III).

Discovery of an ancient MHC category with both class I and class II features.

Two classes of major histocompatibility complex (MHC) molecules, MHC class I and class II, play important roles in our immune system, presenting antigens to functionally distinct T lymphocyte populations. However, the origin of this essential MHC class divergence is poorly understood. Here, we discovered a category of MHC molecules (W-category) in the most primitive jawed vertebrates, cartilaginous fish, and also in bony fish and tetrapods. W-category, surprisingly, possesses class II-type α- and ß-chain organization together with class I-specific sequence motifs for interdomain binding, and the W-category α2 domain shows unprecedented, phylogenetic similarity with ß2-microglobulin of class I. Based on the results, we propose a model in which the ancestral MHC class I molecule evolved from class II-type W-category. The discovery of the ancient MHC group, W-category, sheds a light on the long-standing critical question of the MHC class divergence and suggests that class II type came first.

Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

Immunoglobulin D and its encoding genes: An updated review.

Since the identification of a functional Cδ gene in ostriches, immunoglobulin (Ig) D has been considered to be an extremely evolutionarily conserved Ig isotype besides the IgM found in all classes of jawed vertebrates. However, in contrast to IgM (which remains stable over evolutionary time), IgD shows considerable structural plasticity among vertebrate species and, moreover, its functions are far from elucidated even in humans and mice. Recently, several studies have shown that high expression of the IgD-B-cell receptor (IgD-BCR) may help physiologically autoreactive B cells survive in peripheral lymphoid tissues thanks to unresponsiveness to self-antigens and help their entry into germinal centers to "redeem" autoreactivity via somatic hypermutation. Other studies have demonstrated that secreted IgD may enhance mucosal homeostasis and immunity by linking B cells with basophils to optimize T-helper-2 cell-mediated responses and to constrain IgE-mediated basophil degranulation. Herein, we review the new discoveries on IgD-encoding genes in jawed vertebrates in the past decade. We also highlight advances in the functions of the IgD-BCR and secreted IgD in humans and mice.

A method for making alignments of related protein sequences that share very little similarity; shark interleukin 2 as an example.

An optimized alignment of related protein sequences helps to see their important shared features and to deduce their phylogenetic relationships. At low levels of sequence similarity, there are no suitable computer programs for making the best possible alignment. This review summarizes some guidelines for how in such instances, nevertheless, insightful alignments can be made. The method involves, basically, the understanding of molecular family features at both the protein and intron-exon level, and the collection of many related sequences so that gradual differences may be observed. The method is exemplified by identifying and aligning interleukin 2 (IL-2) and related sequences in Elasmobranchii (sharks/rays) and coelacanth, as other authors have expressed difficulty with their identification. From the point of general immunology, it is interesting that the unusual long "leader" sequence of IL-15, already known in other species, is even more impressively conserved in cartilaginous fish. Furthermore, sequence comparisons suggest that IL-2 in cartilaginous fish has lost its ability to bind an IL-2Rα/15Rα receptor chain, which would prohibit the existence of a mechanism for regulatory T cell regulation identical to mammals.

Generation and preliminary characterization of vertebrate-specific replication-defective Zika virus.

Zika virus (ZIKV) is a mosquito-borne flavivirus that replicates in both vertebrate and insect cells, whereas insect-specific flaviviruses (ISF) replicate only in insect cells. We sought to convert ZIKV, from a dual-tropic flavivirus, into an insect-specific virus for the eventual development of a safe ZIKV vaccine. Reverse genetics was used to introduce specific mutations into the furin cleavage motif within the ZIKV pre-membrane protein (prM). Mutant clones were selected, which replicated well in C6/36 insect cells but exhibited reduced replication in non-human primate (Vero) cells. Further characterization of the furin cleavage site mutants indicated they replicated poorly in both human (HeLa, U251), and baby hamster kidney (BHK-21) cells. One clone with the induced mutation in the prM protein and at positions 291and 452 within the NS3 protein was totally and stably replication-defective in vertebrate cells (VSRD-ZIKV). Preliminary studies in ZIKV sensitive, immunodeficient mice demonstrated that VSRD-ZIKV-infected mice survived and were virus-negative. Our study indicates that a reverse genetic approach targeting the furin cleavage site in prM can be used to select an insect-specific ZIKV with the potential utility as a vaccine strain.

The repertoire of vertebrate STAT transcription factors: Origin and variations in fish.

The stat gene family diversified during early vertebrate evolution thanks to two rounds of whole genome duplication (WGD) to produce a typical repertoire composed of 6 STAT factors (named 1-6). In contrast, only one or two stat genes have been reported in C. elegans and in D. melanogaster. The main types of STAT found from bony fish to mammals are present in Agnathan genomes, but a typical STAT1-6 repertoire is only observed in jawed vertebrates. Comparative syntenies showed that STAT6 was the closest to the ancestor of the family. An extensive survey of stat genes across fish including polyploid species showed that whole genome duplications did not lead to a uniform expansion of stat genes. While 2 to 5 stat1 are present in salmonids, whose genome duplicated about 35My ago, only one copy of stat2 and stat6 is retained. In contrast, common carp, with a recent whole genome duplication (5-10My), possesses a doubled stat repertoire indicating that the elimination of stat2 and stat6 additional copies is not immediate. Altogether our data shed light on the multiplicity of evolutionary pathways followed by key components of the canonical cytokine receptor signalling pathway, and point to differential selective constraints exerted on these factors.

The immune system of jawless vertebrates: insights into the prototype of the adaptive immune system.

Jawless vertebrates diverged from an ancestor of jawed vertebrates approximately 550 million years ago. They mount adaptive immune responses to repetitive antigenic challenges, despite lacking major histocompatibility complex molecules, immunoglobulins, T cell receptors, and recombination-activating genes. Instead of B cell and T cell receptors, agnathan lymphocytes express unique antigen receptors named variable lymphocyte receptors (VLRs), which generate diversity through a gene conversion-like mechanism. Although gnathostome antigen receptors and VLRs are structurally unrelated, jawed and jawless vertebrates share essential features of lymphocyte-based adaptive immunity, including the expression of a single type of receptor on each lymphocyte, clonal expansion of antigen-stimulated lymphocytes, and the dichotomy of cellular and humoral immunity, indicating that the backbone of the adaptive immune system was established in a common ancestor of all vertebrates. Furthermore, recent evidence indicates that, unlike previously thought, agnathans have a unique classical pathway of complement activation where VLRB molecules act as antibodies instead of immunoglobulins. It seems likely that the last common ancestor of all vertebrates had an adaptive immune system resembling that of jawless vertebrates, suggesting that, as opposed to jawed vertebrates, agnathans have retained the prototype of vertebrate adaptive immunity.

Effects of immune challenge on expression of life-history and immune trait expression in sexually reproducing metazoans-a meta-analysis.

BACKGROUND: Life-history theory predicts a trade-off between investment into immune defence and other fitness-related traits. Accordingly, individuals are expected to upregulate their immune response when subjected to immune challenge. However, this is predicted to come at the expense of investment into a range of other traits that are costly to maintain, such as growth, reproduction and survival. Currently, it remains unclear whether the magnitude of such costs, and trade-offs involving immune investment and other traits, manifests consistently across species and sexes. To address this, we conducted a meta-analysis to investigate how changes in sex, ontogenetic stage and environmental factors shape phenotypic trait expression following an immune challenge. RESULTS: We explored the effects of immune challenge on three types of traits across sexually reproducing metazoans: life-history, morphological and proximate immune traits (235 effect sizes, 53 studies, 37 species [21 invertebrates vs. 16 vertebrates]). We report a general negative effect of immune challenge on survival and reproduction, a positive effect on immune trait expression, but no effect on morphology or development time. The negative effects of immune challenge on reproductive traits and survival were larger in females than males. We also report a pronounced effect of the immune treatment agent used (e.g. whether the treatment involved a live pathogen or not) on the host response to immune challenge, and find an effect of mating status on the host response in invertebrates. CONCLUSION: These results suggest that costs associated with immune deployment following an immune challenge are context-dependent and differ consistently in their magnitude across the sexes of diverse taxonomic lineages. We synthesise and discuss the outcomes in the context of evolutionary theory on sex differences in life-history and highlight the need for future studies to carefully consider the design of experiments aimed at disentangling the costs of immune deployment.

Evolution of animal immunity in the light of beneficial symbioses.

Immune system processes serve as the backbone of animal defences against pathogens and thus have evolved under strong selection and coevolutionary dynamics. Most microorganisms that animals encounter, however, are not harmful, and many are actually beneficial. Selection should act on hosts to maintain these associations while preventing exploitation of within-host resources. Here, we consider how several key aspects of beneficial symbiotic associations may shape host immune system evolution. When host immunity is used to regulate symbiont populations, there should be selection to evolve and maintain targeted immune responses that recognize symbionts and suppress but not eliminate symbiont populations. Associating with protective symbionts could relax selection on the maintenance of redundant host-derived immune responses. Alternatively, symbionts could facilitate the evolution of host immune responses if symbiont-conferred protection allows for persistence of host populations that can then adapt. The trajectory of immune system evolution will likely differ based on the type of immunity involved, the symbiont transmission mode and the costs and benefits of immune system function. Overall, the expected influence of beneficial symbiosis on immunity evolution depends on how the host immune system interacts with symbionts, with some interactions leading to constraints while others possibly relax selection on immune system maintenance. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Microbiota-host interactions shape ageing dynamics.

Occupying the interface between host and environment, host-associated microbes play fundamental roles in nutrient absorption, essential metabolite synthesis, development of the immune system, defence against pathogens and pathogenesis. Microbiota composition and function is rather stable during adulthood, while it dramatically changes during early development, frailty and disease. Ageing is associated with progressive decrease of homeostasis, often resulting in disruption of the physiological balance between host and commensal microbes, ultimately leading to dysbiosis and host demise. Generally, high microbial diversity is associated with health and a youthful state, while low individual microbial diversity and larger inter-individual microbial diversity is associated with ageing and disease states. Different species are equipped with species-specific commensal, symbiotic and pathogenic microbial communities. How and whether the specific host-microbiota consortia co-evolved with host physiology to ensure homeostasis and promote individual fitness remains an open question. In this essay, we propose that the evolution of vertebrate-specific immune adaptations may have enabled the establishment of highly diverse, species-specific commensal microbial communities. We discuss how the maintenance of intact immune surveillance mechanisms, which allow discrimination between commensal and pathogenic bacteria, fail during ageing and lead to the onset of known ageing-related diseases. We discuss how host-microbiota interactions are key to maintaining homeostasis despite external perturbations, but also how they affect a range of host-specific ageing-related phenotypes. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Co-evolution of mutagenic genome editors and vertebrate adaptive immunity.

The adaptive immune systems of all vertebrates rely on self-DNA mutating enzymes to assemble their antigen receptors in lymphocytes of their two principal lineages. In jawed vertebrates, the RAG1/2 recombinase directs V(D)J recombination of B cell and T cell receptor genes, whereas the activation-induced cytidine deaminase AID engages in their secondary modification. The recombination activating genes (RAG) 1 and 2 evolved from an ancient transposon-encoded genome modifier into a self-DNA mutator serving adaptive immunity; this was possible as a result of domestication, involving several changes in RAG1 and RAG2 proteins suppressing transposition and instead facilitating-coupled cleavage and recombination. By contrast, recent evidence supports the notion that the antigen receptors of T-like and B-like cells of jawless vertebrates, designated variable lymphocyte receptors (VLRs), are somatically assembled through a process akin to gene conversion that is believed to be dependent on the activities of distant relatives of AID, the cytidine deaminases CDA1 and CDA2, respectively. It appears, therefore, that the precursors of AID and CDAs underwent a domestication process that changed their target range from foreign nucleic acids to self-DNA; this multi-step evolutionary process ensured that the threat to host genome integrity was minimized. Here, we review recent findings illuminating the evolutionary steps associated with the domestication of the two groups of genome editors, RAG1/2 and cytidine deaminases, indicating how they became the driving forces underlying the emergence of vertebrate adaptive immune systems.

Faithful preclinical mouse models for better translation to bedside in the field of immuno-oncology.

The success of immunotherapy using immune checkpoint inhibitors has changed the practice of cancer treatment tremendously. However, there are still many clinical challenges, such as drug resistance, predictive biomarker development, exploration of combination therapies, and prediction of immune-related adverse events in preclinical settings. To overcome these problems, it is essential to establish faithful preclinical mouse models that recapitulate the clinical features, molecular genetics, biological heterogeneity, and immune microenvironment of human cancers. Here we review the advantages and disadvantages of current preclinical mouse models, including syngeneic murine tumor cell lines, autochthonous tumor models, cancer cell line-derived xenografts, patient-derived-xenografts, and various kinds of immunologically humanized mice. We discuss how these models should be characterized and applied in preclinical settings, and how we should prepare preclinical studies for successful translation from bench to bedside.

The Immunoglobulins: New Insights, Implications, and Applications.

Immunoglobulins (Igs), as one of the hallmarks of adaptive immunity, first arose approximately 500 million years ago with the emergence of jawed vertebrates. Two events stand out in the evolutionary history of Igs from cartilaginous fish to mammals: (a) the diversification of Ig heavy chain (IgH) genes, resulting in Ig isotypes or subclasses associated with novel functions, and (b) the diversification of genetic and structural strategies, leading to the creation of the antibody repertoire we know today. This review first gives an overview of the IgH isotypes identified in jawed vertebrates to date and then highlights the implications or applications of five new recent discoveries arising from comparative studies of Igs derived from different vertebrate species.

Lack of statistical power as a major limitation in understanding MHC-mediated immunocompetence in wild vertebrate populations.

Disentangling the sources of variation in developing an effective immune response against pathogens is of major interest to immunoecology and evolutionary biology. To date, the link between immunocompetence and genetic variation at the major histocompatibility complex (MHC) has received little attention in wild animals, despite the key role of MHC genes in activating the adaptive immune system. Although several studies point to a link between MHC and immunocompetence, negative findings have also been reported. Such disparate findings suggest that limited statistical power might be affecting studies on this topic, owing to insufficient sample sizes and/or a generally small effect of MHC on the immunocompetence of wild vertebrates. To clarify this issue, we investigated the link between MHC variation and seven immunocompetence proxies in a large sample of barn owls and estimated the effect sizes and statistical power of this and published studies on this topic. We found that MHC poorly explained variation in immunocompetence of barn owls, with small-to-moderate associations between MHC and immunocompetence in owls (effect size: .1 ≥ r ≤ .3) similar to other vertebrates studied to date. Such small-to-moderate effects were largely associated with insufficient power, which was only sufficient (>0.8) to detect moderate-to-large effect sizes (r ≥ .3). Thus, studies linking MHC variation with immunocompetence in wild populations are underpowered to detect MHC effects, which are likely to be of generally small magnitude. Larger sample sizes (>200) will be required to achieve sufficient power in future studies aiming to robustly test for a link between MHC variation and immunocompetence.

A trade-off model for immunocompetence: The potential contribution of immunological regulation in invasive vertebrate success.

Invasive species have become a prolific environmental issue, second only to climate change, yet many of the phenomena that facilitate invasive success are not well understood (Phillip & Shine, Proc. Roy. Soc. B, 273, 1545-1550). The combination of several generalist life-history traits, certain physiological mechanisms, and environmental conditions is thought to play a significant role in invasion success. The ability to undergo fitness trade-offs-to reallocate nutritional and energetic resources towards processes that increase reproduction, growth, and dispersal-is also thought to be an adaptive quality of many invasive species. Due to their inherent flexibility, phenotypically plastic traits in particular are often targeted for fitness reallocations. Immune function, for example, is determined by a highly plastic phenotype, which is crucial for combating a diverse array of pathogens. When active, immune function also demands extensive resources from the host. Laboratory and field studies suggest that certain aspects of the immune system are more costly than others, though, and that its components can be regulated independent of one another. In invasive species undergoing fitness trade-offs, costly innate inflammatory responses are often downregulated, while antibody-mediated responses may be enhanced. A combination of fixed physiological responses and environmentally induced trade-offs are thought to regulate the immune system, though the relationship between these facets of regulation is still an area of active research. The field of ecoimmunology, then, has emerged in effort to understand the phenomena by which individual immune regulation can drive (and be driven by) species-level ecology and evolution, and therefore be linked to invasive success (Downs et al., 2014. Integr. Compar. Biol., 54, 340-352).