Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by the emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health, and there is an urgent need to develop safe and effective vaccines. Here, we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV-2. We show that our vaccine candidate, GRAd-COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies that neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, T helper (Th)1-dominated cellular response. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV-2-neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at a massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of the GRAd-COV2 vaccine in a currently ongoing phase I clinical trial (ClinicalTrials.gov: NCT04528641).

Detection of Ebola Virus Antibodies in Fecal Samples of Great Apes in Gabon.

Based on a large study conducted on wild great ape fecal samples collected in regions of Gabon where previous human outbreaks of Ebola virus disease have occurred between 1994 and 2002, we provide evidence for prevalence of Zaire ebolavirus (EBOV)-specific antibodies of 3.9% (immunoglobulin G (IgG)) and 3.5% (immunoglobulin M (IgM)) in chimpanzees and 8.8% (IgG) and 2.4% (IgM) in gorillas. Importantly, we observed a high local prevalence (31.2%) of anti-EBOV IgG antibodies in gorilla samples. This high local rate of positivity among wild great apes raises the question of a spatially and temporally localized increase in EBOV exposure risk and the role that can be played by these animals as sentinels of the virus's spread or reemergence in a given area.

Adaptation to Host-Specific Bacterial Pathogens Drives Rapid Evolution of a Human Innate Immune Receptor.

The selective pressure by infectious agents is a major driving force in the evolution of humans and other mammals. Members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family serve as receptors for bacterial pathogens of the genera Haemophilus, Helicobacter, Neisseria, and Moraxella, which engage CEACAMs via distinct surface adhesins. While microbial attachment to epithelial CEACAMs facilitates host colonization, recognition by CEACAM3, a phagocytic receptor expressed by granulocytes, eliminates CEACAM-binding bacteria. Sequence analysis of primate CEACAM3 orthologs reveals that this innate immune receptor is one of the most rapidly evolving human proteins. In particular, the pathogen-binding extracellular domain of CEACAM3 shows a high degree of non-synonymous versus synonymous nucleotide exchanges, indicating an exceptionally strong positive selection. Using CEACAM3 domains derived from different primates, we find that the amino acid alterations found in CEACAM3 translate into characteristic binding patterns for bacterial adhesins. One such amino acid residue is F62 in human and chimp CEACAM3, which is not present in other primates and which is critical for binding the OMP P1 adhesin of Haemophilus aegyptius. Incorporation of the F62-containing motif into gorilla CEACAM3 results in a gain-of-function phenotype with regard to phagocytosis of H. aegyptius. Moreover, CEACAM3 polymorphisms found in human subpopulations widen the spectrum of recognized bacterial adhesins, suggesting an ongoing multivariate selection acting on this innate immune receptor. The species-specific detection of diverse bacterial adhesins helps to explain the exceptionally fast evolution of CEACAM3 within the primate lineage and provides an example of Red Queen dynamics in the human genome.

Discovery of gorilla MHC-C expressing C1 ligand for KIR.

In comparison to humans and chimpanzees, gorillas show low diversity at MHC class I genes (Gogo), as reflected by an overall reduced level of allelic variation as well as the absence of a functionally important sequence motif that interacts with killer cell immunoglobulin-like receptors (KIR). Here, we use recently generated large-scale genomic sequence data for a reassessment of allelic diversity at Gogo-C, the gorilla orthologue of HLA-C. Through the combination of long-range amplifications and long-read sequencing technology, we obtained, among the 35 gorillas reanalyzed, three novel full-length genomic sequences including a coding region sequence that has not been previously described. The newly identified Gogo-C*03:01 allele has a divergent recombinant structure that sets it apart from other Gogo-C alleles. Domain-by-domain phylogenetic analysis shows that Gogo-C*03:01 has segments in common with Gogo-B*07, the additional B-like gene that is present on some gorilla MHC haplotypes. Identified in ~ 50% of the gorillas analyzed, the Gogo-C*03:01 allele exclusively encodes the C1 epitope among Gogo-C allotypes, indicating its important function in controlling natural killer cell (NK cell) responses via KIR. We further explored the hypothesis whether gorillas experienced a selective sweep which may have resulted in a general reduction of the gorilla MHC class I repertoire. Our results provide little support for a selective sweep but rather suggest that the overall low Gogo class I diversity can be best explained by drastic demographic changes gorillas experienced in the ancient and recent past.

Gorilla MHC class I gene and sequence variation in a comparative context.

Comparisons of MHC gene content and diversity among closely related species can provide insights into the evolutionary mechanisms shaping immune system variation. After chimpanzees and bonobos, gorillas are humans' closest living relatives; but in contrast, relatively little is known about the structure and variation of gorilla MHC class I genes (Gogo). Here, we combined long-range amplifications and long-read sequencing technology to analyze full-length MHC class I genes in 35 gorillas. We obtained 50 full-length genomic sequences corresponding to 15 Gogo-A alleles, 4 Gogo-Oko alleles, 21 Gogo-B alleles, and 10 Gogo-C alleles including 19 novel coding region sequences. We identified two previously undetected MHC class I genes related to Gogo-A and Gogo-B, respectively, thereby illustrating the potential of this approach for efficient and highly accurate MHC genotyping. Consistent with their phylogenetic position within the hominid family, individual gorilla MHC haplotypes share characteristics with humans and chimpanzees as well as orangutans suggesting a complex history of the MHC class I genes in humans and the great apes. However, the overall MHC class I diversity appears to be low further supporting the hypothesis that gorillas might have experienced a reduction of their MHC repertoire.

Potent restriction of HIV-1 and SIVmac239 replication by African green monkey TRIM5α.

BACKGROUND: The TRIM5α protein is a principal restriction factor that contributes to an HIV-1 replication block in rhesus macaque CD4+ T cells by preventing reverse transcription. HIV-1 restriction is induced in human CD4+ T cells by expression of rhesus TRIM5α as well as those of other old world monkeys. While TRIM5α restriction has been extensively studied in single-round infection assays, fewer studies have examined restriction after extended viral replication. RESULTS: To examine TRIM5α restriction of replication, we studied the ability of TRIM5α proteins from African green monkey (AgmTRIM5α) and gorilla (gorTRIM5α) to restrict HIV-1 and SIVmac239 replication. These xenogeneic TRIM5α genes were transduced into human Jurkat-CCR5 cells (JR5), which were then exposed to HIV-1 or SIVmac239. In our single-round infection assays, AgmTRIM5α showed a relatively modest 4- to 10-fold restriction of HIV-1 and SIVmac239, while gorTRIM5α produced a 2- and 3-fold restriction of HIV-1 and SIVmac239, respectively, consistent with the majority of previously published single-round studies. To assess the impact of these modest effects on infection, we tested restriction in replication systems initiated with either cell-free or cell-to-cell challenges. AgmTRIM5α powerfully restricted both HIV-1 and SIVmac239 replication 14 days after cell-free infection, with a ≥ 3-log effect. Moreover, expression of AgmTRIM5α restricted HIV-1 and SIVmac239 replication by 2-logs when co-cultured with infected JR5 cells for 12 days. In contrast, neither expression of gorTRIM5α nor rhesus TRIM5α induced significant resistance when co-cultured with infected cells. Follow up experiments showed that the observed differences between replication and infection were not due to assembly defects as xenogeneic TRIM5α expression had no effect on either virion production or specific infectivity. CONCLUSIONS: Our results indicate that AgmTRIM5α has a much greater effect on extended replication than on any single infection event, suggesting that AgmTRIM5α restriction acts cumulatively, building up over many rounds of replication. Furthermore, AgmTRIM5α was able to potently restrict both HIV-1 and SIV replication in a cell-to-cell infection challenge. Thus, AgmTRIM5α is unique among the TRIM5α species tested to date, being able to restrict even at the high multiplicities of infection presented by mixed culture with nonrestrictive infected cells.

Different selective pressures shape the evolution of Toll-like receptors in human and African great ape populations.

The study of the genetic and selective landscape of immunity genes across primates can provide insight into the existing differences in susceptibility to infection observed between human and non-human primates. Here, we explored how selection has driven the evolution of a key family of innate immunity receptors, the Toll-like receptors (TLRs), in African great ape species. We sequenced the 10 TLRs in various populations of chimpanzees and gorillas, and analysed these data jointly with a human data set. We found that purifying selection has been more pervasive in great apes than in humans. Furthermore, in chimpanzees and gorillas, purifying selection has targeted TLRs irrespectively of whether they are endosomal or cell surface, in contrast to humans where strong selective constraints are restricted to endosomal TLRs. These observations suggest important differences in the relative importance of TLR-mediated pathogen sensing, such as that of recognition of flagellated bacteria by TLR5, between humans and great apes. Lastly, we used a population genetics-phylogenetics method that jointly analyses polymorphism and divergence data to detect fine-scale variation in selection pressures at specific codons within TLR genes. We identified different codons at different TLRs as being under positive selection in each species, highlighting that functional variation at these genes has conferred a selective advantage in immunity to infection to specific primate species. Overall, this study showed that the degree of selection driving the evolution of TLRs has largely differed between human and non-human primates, increasing our knowledge on their respective biological contribution to host defence in the natural setting.

Sequencing and comparative analysis of the gorilla MHC genomic sequence.

Major histocompatibility complex (MHC) genes play a critical role in vertebrate immune response and because the MHC is linked to a significant number of auto-immune and other diseases it is of great medical interest. Here we describe the clone-based sequencing and subsequent annotation of the MHC region of the gorilla genome. Because the MHC is subject to extensive variation, both structural and sequence-wise, it is not readily amenable to study in whole genome shotgun sequence such as the recently published gorilla genome. The variation of the MHC also makes it of evolutionary interest and therefore we analyse the sequence in the context of human and chimpanzee. In our comparisons with human and re-annotated chimpanzee MHC sequence we find that gorilla has a trimodular RCCX cluster, versus the reference human bimodular cluster, and additional copies of Class I (pseudo)genes between Gogo-K and Gogo-A (the orthologues of HLA-K and -A). We also find that Gogo-H (and Patr-H) is coding versus the HLA-H pseudogene and, conversely, there is a Gogo-DQB2 pseudogene versus the HLA-DQB2 coding gene. Our analysis, which is freely available through the VEGA genome browser, provides the research community with a comprehensive dataset for comparative and evolutionary research of the MHC.

Noninvasive follow-up of simian immunodeficiency virus infection in wild-living nonhabituated western lowland gorillas in Cameroon.

Simian immunodeficiency viruses infecting western lowland gorillas (SIVgor) are closely related to HIV-1 and are most likely the ancestors of HIV-1 groups O and P. At present, limited data are available on genetic diversity, transmission, viral evolution, and pathogenicity of SIVgor in its natural host. Between 2004 and 2011, 961 putative gorilla fecal samples were collected at the Campo Ma'an National Park, Cameroon. Among them, 16% cross-reacted with HIV-1 antibodies, corresponding to at least 34 infected gorillas. Combining host genotyping and field data, we identified four social groups composed of 7 to 15 individuals each, with SIV rates ranging from 13% to 29%. Eleven SIVgor-infected gorillas were sampled multiple times; two most likely seroconverted during the study period, showing that SIVgor continues to spread. Phylogenetic analysis of partial env and pol sequences revealed cocirculation of closely related and divergent strains among gorillas from the same social group, indicating SIVgor transmissions within and between groups. Parental links could be inferred for some gorillas infected with closely related strains, suggesting vertical transmission, but horizontal transmission by sexual or aggressive behavior was also suspected. Intrahost molecular evolution in one gorilla over a 5-year period showed viral adaptations characteristic of escape mutants, i.e., V1V2 loop elongation and an increased number of glycosylation sites. Here we show for the first time the feasibility of noninvasive monitoring of nonhabituated gorillas to study SIVgor infection over time at both the individual and population levels. This approach can also be applied more generally to study other pathogens in wildlife.

Comparative studies of placentation and immunology in non-human primates suggest a scenario for the evolution of deep trophoblast invasion and an explanation for human pregnancy disorders.

Deep trophoblast invasion in the placental bed has been considered the hallmark of human pregnancy. It occurs by two routes, interstitial and endovascular, and results in transformation of the walls of the spiral arteries as they traverse the decidua and the inner third of the myometrium. Disturbances in this process are associated with reproductive disorders such preeclampsia. In contrast, trophoblast invasion in Old World monkeys occurs only by the endovascular route and seldom reaches the myometrium. Recently, it was shown that this pattern is maintained in gibbons, but that the human arrangement also occurs in chimpanzee and gorilla. There is an interesting parallel with results from placental immunology regarding the evolution of the major histocompatability complex class I antigen HLA-C and its cognate receptors. HLA-C is not present in Old World monkeys or gibbons. It emerged in the orangutan and became polymorphic in the lineage leading to gorilla, bonobo, chimpanzee, and human. Interaction between HLA-C1 and HLA-C2 on the surface of trophoblast and killer immunoglobulin-like receptors (KIRs) expressed by uterine natural killer cells are important regulators of trophoblast invasion. Evolution of this system in great apes may have been one prerequisite for deep trophoblast invasion but seems to have come at a price. The evidence now suggests that certain combinations of maternal genotype for KIRs and fetal genotype for HLA-C imply an increased risk of preeclampsia, fetal growth restriction, and recurrent abortion. The fetal genotype is in part derived from the father providing an explanation for the paternal contribution to reproductive disorders.

NetMHCpan, a method for MHC class I binding prediction beyond humans.

Binding of peptides to major histocompatibility complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC genomic region (called HLA) is extremely polymorphic comprising several thousand alleles, each encoding a distinct MHC molecule. The potentially unique specificity of the majority of HLA alleles that have been identified to date remains uncharacterized. Likewise, only a limited number of chimpanzee and rhesus macaque MHC class I molecules have been characterized experimentally. Here, we present NetMHCpan-2.0, a method that generates quantitative predictions of the affinity of any peptide-MHC class I interaction. NetMHCpan-2.0 has been trained on the hitherto largest set of quantitative MHC binding data available, covering HLA-A and HLA-B, as well as chimpanzee, rhesus macaque, gorilla, and mouse MHC class I molecules. We show that the NetMHCpan-2.0 method can accurately predict binding to uncharacterized HLA molecules, including HLA-C and HLA-G. Moreover, NetMHCpan-2.0 is demonstrated to accurately predict peptide binding to chimpanzee and macaque MHC class I molecules. The power of NetMHCpan-2.0 to guide immunologists in interpreting cellular immune responses in large out-bred populations is demonstrated. Further, we used NetMHCpan-2.0 to predict potential binding peptides for the pig MHC class I molecule SLA-1*0401. Ninety-three percent of the predicted peptides were demonstrated to bind stronger than 500 nM. The high performance of NetMHCpan-2.0 for non-human primates documents the method's ability to provide broad allelic coverage also beyond human MHC molecules. The method is available at http://www.cbs.dtu.dk/services/NetMHCpan.

Ancient haplotypes of the HLA Class II region.

Allelic variation in codons that specify amino acids that line the peptide-binding pockets of HLA's Class II antigen-presenting proteins is superimposed on strikingly few deeply diverged haplotypes. These haplotypes appear to have been evolving almost independently for tens of millions of years. By complete resequencing of 20 haplotypes across the approximately 100-kbp region that spans the HLA-DQA1, -DQB1, and -DRB1 genes, we provide a detailed view of the way in which the genome structure at this locus has been shaped by the interplay of selection, gene-gene interaction, and recombination.

Identification of two pseudogenes with sequence homology to human and gorilla MHC class IA genes: ancestral haplotype in the Filipino population.

While characterizing exons 2 and 3 of the class I human leukocyte antigen (HLA)-A locus in human lymphocytes, two similar but unexpected PCR products were detected in six samples of Filipino ethnicity. A nucleotide sequence analysis of the two amplicons, tentatively named HLA-COQ and HLA-DEL, rendered them as two novel and seemingly related sequences, both with homology to the gorilla and human major histocompatibility complex (MHC) A locus. Exon 2 is similar to the published human pseudogenes HLA-BEL, HLA-Y, and to primate MHC Gogo-A*0501, differing by 2 bp from HLA-BEL, and HLA-Y, and by 4 bp from Gogo-A*0501. Exon 3 is most similar to HLA-A*2902 and A*310102, differing by 7 bp from A*2902, and by 8 bp from A*31012. Genomic sequence comparison of exons 1 to 8 indicates that their closest published match is to the Gogo-A*0501. Complete typing at the HLA-A, -B, -C, DRB1, and DRB5 loci for the six samples yielded the reoccurring types: HLA-A*3401, -B*1521/1525, -Cw*0403, -DRB1*150201, and DRB5*010101. Thus far, HLA-COQ and HLA-DEL have been detected only in Filipino samples containing these HLA types. The HLA-COQ gene is nonfunctional based on a stop codon located in exon 4. HLA-DEL is also a nonfunctional gene because of the dual cytosine insertion in exon 4, with a reading frame shift generating a stop codon downstream. Parsimony analysis of the two pseudogenes with 31 other primate A locus coding regions resulted in a phylogenetic tree that segregated the two pseudogenes with the Gogo-A*0501, suggesting that HLA-COQ, HLA-DEL, and Gogo-A*0501 evolved from a common ancestral allele.

Domain shuffling has been the main mechanism forming new hominoid killer cell Ig-like receptors.

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I-specific receptors, which regulate NK cell responses and are also expressed on subpopulations of T cells. KIR haplotypes vary in gene content, which, in combination with allelic polymorphism, extensively diversifies the KIR genotype both within and between human populations. Species comparison indicates that formation of new KIR genes and loss of old ones are frequent events, so that few genes are conserved even between closely related species. In this regard, the hominoids define a time frame that is particularly informative for understanding the processes of KIR evolution and its potential impact on killer cell biology. KIR cDNA were characterized from PBMC of three gorillas, and genomic DNA were characterized for six additional individuals. Eleven gorilla KIR genes were defined. With attainment of these data, a set of 75 KIR sequences representing five hominoid species was assembled, which also included rhesus monkey, cattle, and rodent KIR. Searching this data set for recombination events, and phylogenetic analysis using Bayesian methods, demonstrated that new KIR were usually the result of recombination between loci in which complete protein domains were shuffled. Further phylogenetic analysis of the KIR sequences after removal of confounding recombined segments showed that only two KIR genes, KIR2DL4 and KIR2DL5, have been preserved throughout hominoid evolution, and one of them, KIR2DL4, is also common to rhesus monkey and hominoids. Other KIR genes represent recombinant forms present in a minority of species, often only one, as exemplified by 8 of the 11 gorilla KIR genes.

Conservation in decay accelerating factor (DAF) structure among primates.

The decay accelerating factor (DAF, CD55) protects self cells from activation of autologous complement on their surfaces. It functions to disable the C3 convertases, the central amplification enzymes of the cascade. Its active site(s) are contained within four approximately 60 amino acid long units, termed complement control protein repeats (CCPs), which are suspended above the cell surface on a 68 amino acid long serine/threonine (S/T)-rich cushion that derives from three exons. We previously proposed a molecular model of human DAF's four CCPs in which certain amino acids were postulated to be recognition sites for the interaction between DAF and the C3 convertases. In the current study, we characterized DAF in five non-human primates: the great apes, gorilla and common chimpanzee, and the Old World monkeys: hamadryas baboon, Rhesus macaque, and patas monkey. Amino acid homology to human DAF was approximately 98% for the two great apes and 83% for the three Old World monkeys. The above cited putative ligand interactive residues were found to be fully conserved in all of the non-human primates, although there were amino acid changes outside of these areas. In the chimpanzee, alternative splicing of the S/T region was found potentially to be the source of multiple protein isoforms in erythrocytes, whereas in the patas monkey, similar alternative splicing was observed but only one protein band was seen. Interestingly, a Rhesus macaque was found to exhibit a phenomenon paralleling the human Cromer Dr(a-) blood group, in which a 44-base pair deletion in CCP3 leads to a frameshift and early STOP codon.

Nomenclature of immunoglobulin A and other proteins of the mucosal immune system. IUIS/WHO Subcommittee on IgA Nomenclature.

The predominant immunoglobulin found in exocrine secretions of humans and most other mammals is secretory IgA, a polymeric form of IgA containing an additional glycoprotein chain designated "secretory component". In this article recommended abbreviations are proposed for the following forms of human IgA and other proteins of related interest: secretory IgA, secretory IgM, secretory component, polymeric immunoglobulin receptor, polymeric IgA, monomeric IgA, IgA subclass 1, IgA subclass, 2, A2 allotype marker 1, and A2 allotype marker 2.

Characterization of a macaque anti-Rh17-like monoclonal antibody.

In order to produce macaque monoclonal antibodies (mAbs) against human red blood cell (RBC) antigens, macaques were immunized with human and gorilla RBCs and their spleen lymphocytes were fused with man-mouse heteromyeloma cells. One macaque-mouse heterohybridoma produced a macaque IgGx (Cvn2-4D5) which agglutinated all human RBCs but not rare human variants Dc-,D-, and Rhnull. Thus, Cyn2-4D5 exhibited RH17-like reactivity. The specificity of Cyn2-4D5 for RHCE-encoded polypeptides was confirmed by specific immunoprecipitation of RhcE and RhCe polypeptides from human RBCs and the absence of immunoprecipitation of the RhD polypeptides extracted from D-RBCs. This study demonstrates that it is possible to produce macaque mAbs against human RBC blood group antigens.