The KIR repertoire of a West African chimpanzee population is characterized by limited gene, allele, and haplotype variation.

Introduction: The killer cell immunoglobulin-like receptors (KIR) play a pivotal role in modulating the NK cell responses, for instance, through interaction with major histocompatibility complex (MHC) class I molecules. Both gene systems map to different chromosomes but co-evolved during evolution. The human KIR gene family is characterized by abundant allelic polymorphism and copy number variation. In contrast, our knowledge of the KIR repertoire in chimpanzees is limited to 39 reported alleles, with no available population data. Only three genomic KIR region configurations have been mapped, and seventeen additional ones were deduced by genotyping. Methods: Previously, we documented that the chimpanzee MHC class I repertoire has been skewed due to an ancient selective sweep. To understand the depth of the sweep, we set out to determine the full-length KIR transcriptome - in our MHC characterized pedigreed West African chimpanzee cohort - using SMRT sequencing (PacBio). In addition, the genomic organization of 14 KIR haplotypes was characterized by applying a Cas9-mediated enrichment approach in concert with long-read sequencing by Oxford Nanopore Technologies. Results: In the cohort, we discovered 35 undescribed and 15 already recorded Patr-KIR alleles, and a novel hybrid KIR gene. Some KIR transcripts are subject to evolutionary conserved alternative splicing events. A detailed insight on the KIR region dynamics (location and order of genes) was obtained, however, only five new KIR region configurations were detected. The population data allowed to investigate the distribution of the MHC-C1 and C2-epitope specificity of the inhibitory lineage III KIR repertoire, and appears to be skewed towards C2. Discussion: Although the KIR region is known to evolve fast, as observed in other primate species, our overall conclusion is that the genomic architecture and repertoire in West African chimpanzees exhibit only limited to moderate levels of variation. Hence, the ancient selective sweep that affected the chimpanzee MHC class I region may also have impacted the KIR system.

Longitudinal positron emission tomography and postmortem analysis reveals widespread neuroinflammation in SARS-CoV-2 infected rhesus macaques.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients initially develop respiratory symptoms, but they may also suffer from neurological symptoms. People with long-lasting effects after acute infections with severe respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., post-COVID syndrome or long COVID, may experience a variety of neurological manifestations. Although we do not fully understand how SARS-CoV-2 affects the brain, neuroinflammation likely plays a role. METHODS: To investigate neuroinflammatory processes longitudinally after SARS-CoV-2 infection, four experimentally SARS-CoV-2 infected rhesus macaques were monitored for 7 weeks with 18-kDa translocator protein (TSPO) positron emission tomography (PET) using [18F]DPA714, together with computed tomography (CT). The baseline scan was compared to weekly PET-CTs obtained post-infection (pi). Brain tissue was collected following euthanasia (50 days pi) to correlate the PET signal with TSPO expression, and glial and endothelial cell markers. Expression of these markers was compared to brain tissue from uninfected animals of comparable age, allowing the examination of the contribution of these cells to the neuroinflammatory response following SARS-CoV-2 infection. RESULTS: TSPO PET revealed an increased tracer uptake throughout the brain of all infected animals already from the first scan obtained post-infection (day 2), which increased to approximately twofold until day 30 pi. Postmortem immunohistochemical analysis of the hippocampus and pons showed TSPO expression in cells expressing ionized calcium-binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and collagen IV. In the hippocampus of SARS-CoV-2 infected animals the TSPO+ area and number of TSPO+ cells were significantly increased compared to control animals. This increase was not cell type specific, since both the number of IBA1+TSPO+ and GFAP+TSPO+ cells was increased, as well as the TSPO+ area within collagen IV+ blood vessels. CONCLUSIONS: This study manifests [18F]DPA714 as a powerful radiotracer to visualize SARS-CoV-2 induced neuroinflammation. The increased uptake of [18F]DPA714 over time implies an active neuroinflammatory response following SARS-CoV-2 infection. This inflammatory signal coincides with an increased number of TSPO expressing cells, including glial and endothelial cells, suggesting neuroinflammation and vascular dysregulation. These results demonstrate the long-term neuroinflammatory response following a mild SARS-CoV-2 infection, which potentially precedes long-lasting neurological symptoms.

Brain Inflammation and Intracellular α-Synuclein Aggregates in Macaques after SARS-CoV-2 Infection.

SARS-CoV-2 causes acute respiratory disease, but many patients also experience neurological complications. Neuropathological changes with pronounced neuroinflammation have been described in individuals after lethal COVID-19, as well as in the CSF of hospitalized patients with neurological complications. To assess whether neuropathological changes can occur after a SARS-CoV-2 infection, leading to mild-to-moderate disease, we investigated the brains of four rhesus and four cynomolgus macaques after pulmonary disease and without overt clinical symptoms. Postmortem analysis demonstrated the infiltration of T-cells and activated microglia in the parenchyma of all infected animals, even in the absence of viral antigen or RNA. Moreover, intracellular α-synuclein aggregates were found in the brains of both macaque species. The heterogeneity of these manifestations in the brains indicates the virus' neuropathological potential and should be considered a warning for long-term health risks, following SARS-CoV-2 infection.

Comparative genetics of KIR haplotype diversity in humans and rhesus macaques: the balancing act.

The role of natural killer (NK) cells is tightly modulated by interactions of killer cell immunoglobulin-like receptors (KIR) with their ligands of the MHC class I family. Several characteristics of the KIR gene products are conserved in primate evolution, like the receptor structures and the variegated expression pattern. At the genomic level, however, the clusters encoding the KIR family display species-specific diversity, reflected by differential gene expansions and haplotype architecture. The human KIR cluster is extensively studied in large cohorts from various populations, which revealed two KIR haplotype groups, A and B, that represent more inhibitory and more activating functional profiles, respectively. So far, genomic KIR analyses in large outbred populations of non-human primate species are lacking. In this study, we roughly quadrupled the number of rhesus macaques studied for their KIR transcriptome (n = 298). Using segregation analysis, we defined 112 unique KIR region configurations, half of which display a more inhibitory profile, whereas the other half has a more activating potential. The frequencies and functional potential of these profiles might mirror the human KIR haplotype groups. However, whereas the human group A and B KIR haplotypes are confined to largely fixed organizations, the haplotypes in macaques feature highly variable gene content. Moreover, KIR homozygosity was hardly encountered in this panel of macaques. This study exhibits highly diverse haplotype architectures in humans and macaques, which nevertheless might have an equivalent effect on the modulation of NK cell activity.

Dynamic evolution of Mhc haplotypes in cynomolgus macaques of different geographic origins.

The major histocompatibility complex (MHC) plays a key role in immune defense, and the Mhc genes of cynomolgus macaque display a high degree of polymorphism. Based on their geographic distribution, different populations of cynomolgus macaques are recognized. Here we present the characterization of the Mhc class I and II repertoire of a large pedigreed group of cynomolgus macaques originating from the mainland north of the isthmus of Kra (N = 42). Segregation analyses resulted in the definition of 81 unreported Mafa-A/B/DRB/DQ/DP haplotypes, which include 32 previously unknown DRB regions. In addition, we report 13 newly defined Mafa-A/B/DRB/DQ/DP haplotypes in a group of cynomolgus macaques originating from the mainland south of the isthmus of Kra/Maritime Southeast Asia (N = 16). A relatively high level of sharing of Mafa-A (51%) and Mafa-B (40%) lineage groups is observed between the populations native to the north and the south of isthmus of Kra. At the allelic level, however, the Mafa-A/B haplotypes seem to be characteristic of a population. An overall comparison of all currently known data revealed that each geographic population has its own specific combinations of Mhc class I and II haplotypes. This illustrates the dynamic evolution of the cynomolgus macaque Mhc region, which was most likely generated by recombination and maintained by selection due to the differential pathogenic pressures encountered in different geographic areas.

Medical imaging of pulmonary disease in SARS-CoV-2-exposed non-human primates.

Chest X-ray (CXR), computed tomography (CT), and positron emission tomography-computed tomography (PET-CT) are noninvasive imaging techniques widely used in human and veterinary pulmonary research and medicine. These techniques have recently been applied in studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-exposed non-human primates (NHPs) to complement virological assessments with meaningful translational readouts of lung disease. Our review of the literature indicates that medical imaging of SARS-CoV-2-exposed NHPs enables high-resolution qualitative and quantitative characterization of disease otherwise clinically invisible and potentially provides user-independent and unbiased evaluation of medical countermeasures (MCMs). However, we also found high variability in image acquisition and analysis protocols among studies. These findings uncover an urgent need to improve standardization and ensure direct comparability across studies.

The Genomic Organization of the LILR Region Remained Largely Conserved Throughout Primate Evolution: Implications for Health And Disease.

The genes of the leukocyte immunoglobulin-like receptor (LILR) family map to the leukocyte receptor complex (LRC) on chromosome 19, and consist of both activating and inhibiting entities. These receptors are often involved in regulating immune responses, and are considered to play a role in health and disease. The human LILR region and evolutionary equivalents in some rodent and bird species have been thoroughly characterized. In non-human primates, the LILR region is annotated, but a thorough comparison between humans and non-human primates has not yet been documented. Therefore, it was decided to undertake a comprehensive comparison of the human and non-human primate LILR region at the genomic level. During primate evolution the organization of the LILR region remained largely conserved. One major exception, however, is provided by the common marmoset, a New World monkey species, which seems to feature a substantial contraction of the number of LILR genes in both the centromeric and the telomeric region. Furthermore, genomic analysis revealed that the killer-cell immunoglobulin-like receptor gene KIR3DX1, which maps in the LILR region, features one copy in humans and great ape species. A second copy, which might have been introduced by a duplication event, was observed in the lesser apes, and in Old and New World monkey species. The highly conserved gene organization allowed us to standardize the LILR gene nomenclature for non-human primate species, and implies that most of the receptors encoded by these genes likely fulfill highly preserved functions.

Rapid Characterization of Complex Killer Cell Immunoglobulin-Like Receptor (KIR) Regions Using Cas9 Enrichment and Nanopore Sequencing.

Long-read sequencing approaches have considerably improved the quality and contiguity of genome assemblies. Such platforms bear the potential to resolve even extremely complex regions, such as multigenic immune families and repetitive stretches of DNA. Deep sequencing coverage, however, is required to overcome low nucleotide accuracy, especially in regions with high homopolymer density, copy number variation, and sequence similarity, such as the MHC and KIR gene clusters of the immune system. Therefore, we have adapted a targeted enrichment protocol in combination with long-read sequencing to efficiently annotate complex KIR gene regions. Using Cas9 endonuclease activity, segments of the KIR gene cluster were enriched and sequenced on an Oxford Nanopore Technologies platform. This provided sufficient coverage to accurately resolve and phase highly complex KIR haplotypes. Our strategy eliminates PCR-induced amplification errors, facilitates rapid characterization of large and complex multigenic regions, including its epigenetic footprint, and is applicable in multiple species, even in the absence of a reference genome.

The Post-Acute Phase of SARS-CoV-2 Infection in Two Macaque Species Is Associated with Signs of Ongoing Virus Replication and Pathology in Pulmonary and Extrapulmonary Tissues.

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.

Two Human Monoclonal HLA-Reactive Antibodies Cross-React with Mamu-B*008, a Rhesus Macaque MHC Allotype Associated with Control of Simian Immunodeficiency Virus Replication.

MHC class I molecules play an important role in adaptive immune responses against intracellular pathogens. These molecules are highly polymorphic, and many allotypes have been characterized. In a transplantation setting, a mismatch between MHC allotypes may initiate an alloimmune response. Rhesus macaques (Macaca mulatta, Mamu) are valuable as a preclinical model species in transplantation research as well as to evaluate the safety and efficacy of vaccine candidates. In both lines of research, the availability of nonhuman primate MHC-reactive mAbs may enable in vitro monitoring and detection of presence of particular Mamu molecules. In this study, we screened a collection of thoroughly characterized HLA class I-specific human mAbs for cross-reactivity with rhesus macaque MHC class I allotypes. Two mAbs, OK4F9 and OK4F10, recognize an epitope that is defined by isoleucine (I) at amino acid position 142 that is present on the Indian rhesus macaque Mamu-B*008:01 allotype, which is an allotype known to be associated with elite control of SIV replication. The reactive pattern of a third mAb, MUS4H4, is more complex and includes an epitope shared on Mamu-A2*05:01 and -B*001:01-encoded Ags. This is the first description, to our knowledge, of human HLA-reactive mAbs that can recognize Mamu allotypes, and these can be useful tools for in vitro monitoring the presence of the relevant allelic products. Moreover, OK4F9 and OK4F10 can be powerful mAbs for application in SIV-related research.

The Genetic Mechanisms Driving Diversification of the KIR Gene Cluster in Primates.

The activity and function of natural killer (NK) cells are modulated through the interactions of multiple receptor families, of which some recognize MHC class I molecules. The high level of MHC class I polymorphism requires their ligands either to interact with conserved epitopes, as is utilized by the NKG2A receptor family, or to co-evolve with the MHC class I allelic variation, which task is taken up by the killer cell immunoglobulin-like receptor (KIR) family. Multiple molecular mechanisms are responsible for the diversification of the KIR gene system, and include abundant chromosomal recombination, high mutation rates, alternative splicing, and variegated expression. The combination of these genetic mechanisms generates a compound array of diversity as is reflected by the contraction and expansion of KIR haplotypes, frequent birth of fusion genes, allelic polymorphism, structurally distinct isoforms, and variegated expression, which is in contrast to the mainly allelic nature of MHC class I polymorphism in humans. A comparison of the thoroughly studied human and macaque KIR gene repertoires demonstrates a similar evolutionarily conserved toolbox, through which selective forces drove and maintained the diversified nature of the KIR gene cluster. This hypothesis is further supported by the comparative genetics of KIR haplotypes and genes in other primate species. The complex nature of the KIR gene system has an impact upon the education, activity, and function of NK cells in coherence with an individual's MHC class I repertoire and pathogenic encounters. Although selection operates on an individual, the continuous diversification of the KIR gene system in primates might protect populations against evolving pathogens.

Similar patterns of genetic diversity and linkage disequilibrium in Western chimpanzees (Pan troglodytes verus) and humans indicate highly conserved mechanisms of MHC molecular evolution.

BACKGROUND: Many species are threatened with extinction as their population sizes decrease with changing environments or face novel pathogenic threats. A reduction of genetic diversity at major histocompatibility complex (MHC) genes may have dramatic effects on populations' survival, as these genes play a key role in adaptive immunity. This might be the case for chimpanzees, the MHC genes of which reveal signatures of an ancient selective sweep likely due to a viral epidemic that reduced their population size a few million years ago. To better assess how this past event affected MHC variation in chimpanzees compared to humans, we analysed several indexes of genetic diversity and linkage disequilibrium across seven MHC genes on four cohorts of chimpanzees and we compared them to those estimated at orthologous HLA genes in a large set of human populations. RESULTS: Interestingly, the analyses uncovered similar patterns of both molecular diversity and linkage disequilibrium across the seven MHC genes in chimpanzees and humans. Indeed, in both species the greatest allelic richness and heterozygosity were found at loci A, B, C and DRB1, the greatest nucleotide diversity at loci DRB1, DQA1 and DQB1, and both significant global linkage disequilibrium and the greatest proportions of haplotypes in linkage disequilibrium were observed at pairs DQA1 ~ DQB1, DQA1 ~ DRB1, DQB1 ~ DRB1 and B ~ C. Our results also showed that, despite some differences among loci, the levels of genetic diversity and linkage disequilibrium observed in contemporary chimpanzees were globally similar to those estimated in small isolated human populations, in contrast to significant differences compared to large populations. CONCLUSIONS: We conclude, first, that highly conserved mechanisms shaped the diversity of orthologous MHC genes in chimpanzees and humans. Furthermore, our findings support the hypothesis that an ancient demographic decline affecting the chimpanzee populations - like that ascribed to a viral epidemic - exerted a substantial effect on the molecular diversity of their MHC genes, albeit not more pronounced than that experienced by HLA genes in human populations that underwent rapid genetic drift during humans' peopling history. We thus propose a model where chimpanzees' MHC genes regenerated molecular variation through recombination/gene conversion and/or balancing selection after the selective sweep.

Comparative genetics of the major histocompatibility complex in humans and nonhuman primates.

The major histocompatibility complex (MHC) is one of the most gene-dense regions of the mammalian genome. Multiple genes within the human MHC (HLA) show extensive polymorphism, and currently, more than 26,000 alleles divided over 39 different genes are known. Nonhuman primate (NHP) species are grouped into great and lesser apes and Old and New World monkeys, and their MHC is studied mostly because of their important role as animal models in preclinical research or in connection with conservation biology purposes. The evolutionary equivalents of many of the HLA genes are present in NHP species, and these genes may also show abundant levels of polymorphism. This review is intended to provide a comprehensive comparison relating to the organization and polymorphism of human and NHP MHC regions.

Unparalleled Rapid Evolution of KIR Genes in Rhesus and Cynomolgus Macaque Populations.

The killer cell Ig-like receptors (KIR) modulate immune responses through interactions with MHC class I molecules. The KIR region in large cohorts of rhesus and cynomolgus macaque populations were characterized, and the experimental design enabled the definition of a considerable number of alleles (n = 576) and haplotypes, which are highly variable with regard to architecture. Although high levels of polymorphism were recorded, only a few alleles are shared between species and populations. The rapid evolution of allelic polymorphism, accumulated by point mutations, was further confirmed by the emergence of a novel KIR allele in a rhesus macaque family. In addition to allelic variation, abundant orthologous and species-specific KIR genes were identified, the latter of which are frequently generated by fusion events. The concerted action of both genetic mechanisms, in combination with differential selective pressures at the population level, resulted in the unparalleled rapid evolution of the KIR gene region in two closely related macaque species. The variation of the KIR gene repertoire at the species and population level might have an impact on the outcome of preclinical studies with macaque models.

The HLA A03 Supertype and Several Pan Species Major Histocompatibility Complex Class I A Allotypes Share a Preference for Binding Positively Charged Residues in the F Pocket: Implications for Controlling Retroviral Infections.

The major histocompatibility complex (MHC) class I region of humans, chimpanzees (Pan troglodytes), and bonobos (Pan paniscus) is highly similar, and orthologues of HLA-A, -B, and -C are present in both Pan species. Based on functional characteristics, the different HLA-A allotypes are classified into different supertypes. One of them, the HLA A03 supertype, is widely distributed among different human populations. All contemporary known chimpanzee and bonobo MHC class I A allotypes cluster genetically into one of the six HLA-A families, the HLA-A1/A3/A11/A30 family. We report here that the peptide-binding motif of the Patr-A*05:01 allotype, which is commonly present in a cohort of western African chimpanzees, has a strong preference for binding peptides with basic amino acids at the carboxyl terminus. This phenomenon is shared with the family members of the HLA A03 supertype. Based on the chemical similarities in the peptide-binding pocket, we inferred that the preference for binding peptides with basic amino acids at the carboxyl terminus is widely present among the human, chimpanzee, and bonobo MHC-A allotypes. Subsequent in silico peptide-binding predictions illustrated that these allotypes have the capacity to target conserved parts of the proteome of human immunodeficiency virus type 1 (HIV-1) and the simian immunodeficiency virus SIVcpz.IMPORTANCE Most experimentally infected chimpanzees seem to control an HIV-1 infection and are therefore considered to be relatively resistant to developing AIDS. Contemporary free-ranging chimpanzees may carry SIVcpz, and there is evidence for AIDS-like symptoms in these free-ranging animals, whereas SIV infections in bonobos appear to be absent. In humans, the natural control of an HIV-1 infection is strongly associated with the presence of particular HLA class I allotypes. The ancestor of the contemporary living chimpanzees and bonobos survived a selective sweep targeting the MHC class I repertoire. We have put forward a hypothesis that this may have been caused by an ancestral retroviral infection similar to SIVcpz. Characterization of the relevant MHC allotypes may contribute to understanding the shaping of their immune repertoire. The abundant presence of MHC-A allotypes that prefer peptides with basic amino acids at the C termini suggests that these molecules may contribute to the control of retroviral infections in humans, chimpanzees, and bonobos.

Nomenclature report 2019: major histocompatibility complex genes and alleles of Great and Small Ape and Old and New World monkey species.

The major histocompatibility complex (MHC) is central to the innate and adaptive immune responses of jawed vertebrates. Characteristic of the MHC are high gene density, gene copy number variation, and allelic polymorphism. Because apes and monkeys are the closest living relatives of humans, the MHCs of these non-human primates (NHP) are studied in depth in the context of evolution, biomedicine, and conservation biology. The Immuno Polymorphism Database (IPD)-MHC NHP Database (IPD-MHC NHP), which curates MHC data of great and small apes, as well as Old and New World monkeys, has been upgraded. The curators of the database are responsible for providing official designations for newly discovered alleles. This nomenclature report updates the 2012 report, and summarizes important nomenclature issues and relevant novel features of the IPD-MHC NHP Database.

Nomenclature report for killer-cell immunoglobulin-like receptors (KIR) in macaque species: new genes/alleles, renaming recombinant entities and IPD-NHKIR updates.

The Killer-cell Immunoglobulin-like Receptors (KIR) are encoded by a diverse group of genes, which are characterized by allelic polymorphism, gene duplications, and recombinations, which may generate recombinant entities. The number of reported macaque KIR sequences is steadily increasing, and these data illustrate a gene system that may match or exceed the complexity of the human KIR cluster. This report lists the names of quality controlled and annotated KIR genes/alleles with all the relevant references for two different macaque species: rhesus and cynomolgus macaques. Numerous recombinant KIR genes in these species necessitate a revision of some of the earlier-published nomenclature guidelines. In addition, this report summarizes the latest information on the Immuno Polymorphism Database (IPD)-NHKIR Database, which contains annotated KIR sequences from four non-human primate species.

Correction to: Nomenclature report 2019: major histocompatibility complex genes and alleles of great and small ape and old and new world monkey species.

The original version of this article contained a spelling error in the Acknowledgments regarding the name of the funding organisation supporting GM and JAH. UKRI-BBSCR should have been UKRI-BBSRC, as is now indicated correctly below.