Unprecedented polymorphism of Mhc-DRB region configurations in rhesus macaques

The rhesus macaque is an important model in preclinical transplantation research and for the study of chronic and infectious diseases, and so extensive knowledge of its MHC (MhcMamu) is needed. Nucleotide sequencing of exon 2 allowed the detection of 68 Mamu-DRB alleles. Although most alleles belong to loci/lineages that have human equivalents, identical Mhc-DRB alleles are not shared between humans and rhesus macaques. The number of -DRB genes present per haplotype can vary from two to seven in the rhesus macaque, whereas it ranges from one to four in humans. Within a panel of 210 rhesus macaques, 24 Mamu-DRB region configurations can be distinguished differing in the number and composition of loci. None of the Mamu-DRB region configurations has been described for any other species, and only one of them displays major allelic variation giving rise to a total of 33 Mamu-DRB haplotypes. In the human population, only five HLA-DRB region configurations were defined, which in contrast to the rhesus macaque exhibit extensive allelic polymorphism. In comparison with humans, the unprecedented polymorphism of the Mamu-DRB region configurations may reflect an alternative strategy of this primate species to cope with pathogens. Because of the Mamu-DRB diversity, nonhuman primate colonies used for immunological research should be thoroughly typed to facilitate proper interpretation of results. This approach will minimize as well the number of animals necessary to conduct experiments.     

The major histocompatibility complex class II alleles Mamu-DRB1*1003 and -DRB1*0306 are enriched in a cohort of simian immunodeficiency virus-infected rhesus macaque elite controllers

The role of CD4(+) T cells in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication is not well understood. Even though strong HIV- and SIV-specific CD4(+) T-cell responses have been detected in individuals that control viral replication, major histocompatibility complex class II (MHC-II) molecules have not been definitively linked with slow disease progression. In a cohort of 196 SIVmac239-infected Indian rhesus macaques, a group of macaques controlled viral replication to less than 1,000 viral RNA copies/ml. These elite controllers (ECs) mounted a broad SIV-specific CD4(+) T-cell response. Here, we describe five macaque MHC-II alleles (Mamu-DRB*w606, -DRB*w2104, -DRB1*0306, -DRB1*1003, and -DPB1*06) that restricted six SIV-specific CD4(+) T-cell epitopes in ECs and report the first association between specific MHC-II alleles and elite control. Interestingly, the macaque MHC-II alleles, Mamu-DRB1*1003 and -DRB1*0306, were enriched in this EC group (P values of 0.02 and 0.05, respectively). Additionally, Mamu-B*17-positive SIV-infected rhesus macaques that also expressed these two MHC-II alleles had significantly lower viral loads than Mamu-B*17-positive animals that did not express Mamu-DRB1*1003 and -DRB1*0306 (P value of <0.0001). The study of MHC-II alleles in macaques that control viral replication could improve our understanding of the role of CD4(+) T cells in suppressing HIV/SIV replication and further our understanding of HIV vaccine design.     

Molecular determinants of peptide binding to two common rhesus macaque major histocompatibility complex class II molecules

Major histocompatibility complex class II molecules encoded by two common rhesus macaque alleles Mamu-DRB1*0406 and Mamu-DRB*w201 have been purified, and quantitative binding assays have been established. The structural requirements for peptide binding to each molecule were characterized by testing panels of single-substitution analogs of the two previously defined epitopes HIV Env242 (Mamu-DRB1*0406 restricted) and HIV Env482 (Mamu-DRB*w201 restricted). Anchor positions of both macaque DR molecules were spaced following a position 1 (P1), P4, P6, P7, and P9 pattern. The specific binding motif associated with each molecule was distinct, but largely overlapping, and was based on crucial roles of aromatic and/or hydrophobic residues at P1, P6, and P9. Based on these results, a tentative Mamu class II DR supermotif was defined. This pattern is remarkably similar to a previously defined human HLA-DR supermotif. Similarities in binding motifs between human HLA and macaque Mamu-DR molecules were further illustrated by testing a panel of more than 60 different single-substitution analogs of the HLA-DR-restricted HA 307-319 epitope for binding to Mamu-DRB*w201 and HLA-DRB1*0101. The Mamu-DRB1*0406 and -DRB*w201 binding capacity of a set of 311 overlapping peptides spanning the entire simian immunodeficiency virus (SIV) genome was also evaluated. Ten peptides capable of binding both molecules were identified, together with 19 DRB1*0406 and 43 DRB*w201 selective binders. The Mamu-DR supermotif was found to be present in about 75% of the good binders and in 50% of peptides binding with intermediate affinity but only in approximately 25% of the peptides which did not bind either Mamu class II molecule. Finally, using flow cytometric detection of antigen-induced intracellular gamma interferon, we identify a new CD4(+) T-lymphocyte epitope encoded within the Rev protein of SIV.     

Multilocus definition of MHC haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis)

Cynomolgus macaques (Macaca fascicularis) are used widely in biomedical research, and the genetics of their MHC (Mhc-Mafa) has become the focus of considerable attention in recent years. The cohort of Indonesian pedigreed macaques that we present here was typed for Mafa-A, -B, and -DR, by sequencing, as described in earlier studies. Additionally, the DRB region of these animals was characterised by microsatellite analyses. In this study, full-length sequencing of Mafa-DPA/B and -DQA/B in these animals was performed. A total of 75 different alleles were observed; 22 of which have not previously been reported, plus 18 extended exon 2 alleles that were already known. Furthermore, two microsatellites, D6S2854 and D6S2859, were used to characterise the complex Mafa-A region. Sequencing and segregation analyses revealed that the length patterns of these microsatellites are unique for each Mafa-A haplotype. In this work, we present a pedigreed colony of approximately 120 cynomolgus macaques; all of which are typed for the most significant polymorphic MHC class I and class II markers. Offspring of these pedigreed animals are easily characterised for their MHC by microsatellite analyses on the Mafa-A and -DRB regions, which makes the cumbersome sequencing analyses redundant.     

MHC class I characterization of Indonesian cynomolgus macaques

Cynomolgus macaques (Macaca fascicularis) are quickly becoming a useful model for infectious disease and transplantation research. Even though cynomolgus macaques from different geographic regions are used for these studies, there has been limited characterization of full-length major histocompatibility complex (MHC) class I immunogenetics of distinct geographic populations. Here, we identified 48 MHC class I cDNA nucleotide sequences in eleven Indonesian cynomolgus macaques, including 41 novel Mafa-A and Mafa-B sequences. We found seven MHC class I sequences in Indonesian macaques that were identical to MHC class I sequences identified in Malaysian or Mauritian macaques. Sharing of nucleotide sequences between these geographically distinct populations is also consistent with the hypothesis that Indonesia was a source of the Mauritian macaque population. In addition, we found that the Indonesian cDNA sequence Mafa-B7601 is identical throughout its peptide binding domain to Mamu-B03, an allele that has been associated with control of Simian immunodeficiency virus (SIV) viremia in Indian rhesus macaques. Overall, a better understanding of the MHC class I alleles present in Indonesian cynomolgus macaques improves their value as a model for disease research, and it better defines the biogeography of cynomolgus macaques throughout Southeast Asia.     

中华菊头蝠MHCⅡ-DRB 基因遗传多态性及进化

采样自云南同一种群的中华菊头蝠共16 个个体,用于DRB 基因的分子进化和多态性研究。利用翼膜组织提取DNA 基因组,并PCR 克隆测序分析。获得了相差3 bp 的两种不同长度序列类型,A 序列类型263 bp,在研究群体中有15 个等位基因;B 序列类型260 bp,在研究群体中有8 个等位基因。在分析的74 个氨基酸变异位点上检测到12 个正向选择位点。在9 个个体中检测到分布频率最高的等位基因,也有多个等位基因只存在一个个体中。单个个体中最多存在6 个等位基因。遗传多态性分析表明中华菊头蝠DRB 基因具有较高的多态性。中华菊头蝠DRB 基因可能至少存在3 个重复座位。利用已发表的翼手目DRB 第二外显子序列构建的系统进化树表明中华菊头蝠MHCⅡ-DRB 基因处于独立进化支。     

猕猴MHC-DPB1基因外显子2多态性研究

猕猴(Macaca mulatta)是最理想的医学实验灵长类动物, 且为国家二级保护动物。为了解中国猕猴主要组织相容复合体(Major histocompatibility complex, MHC)基因的遗传多态性背景, 为它们在生物医学研究中的应用及其遗传资源的保护提供一定的科学依据, 文章采用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)和克隆测序技术分析了106个四川野生猕猴MHC-DPB1基因的exon 2, 共检测到21个Mamu-DPB1等位基因, 其中有15个为本研究中首次发现的新等位基因; 从整个大的猕猴群体(106个个体)来看, 等位基因频率最高的是Mamu-DPB1*30(0.1120); 单独从不同地理群体来看, 最高等位基因频率分别为: 小金-DPB1*30 (0.1120), 黑水-DPB1*04 (0.1702), 巴中-DPB1*32 (0.1613), 汉源-DPB1*30(0.1120), 九龙-DPB1*04(0.1139); 氨基酸序列比对发现, 猕猴Mamu-DPB1等位基因编码的氨基酸序列中, 有12个氨基酸残基变异位点表现出物种特异性, 其中有9个位于新发现的15个Mamu-DPB1等位基因氨基酸序列中; 不同物种来源的DPB1等位基因系统发生树表明, 猕猴与其近缘物种食蟹猴(Macaca fascicularis)的DPB1等位基因间存在着跨种多态(Trans-species polymorphism)现象。研究还表明, MHC-DPB1等位基因在中国猕猴群体和先前为主要研究对象的印度猕猴群体间具有较大的差异。     

MHC class I A region diversity and polymorphism in macaque species

The HLA-A locus represents a single copy gene that displays abundant allelic polymorphism in the human population, whereas, in contrast, a nonhuman primate species such as the rhesus macaque (Macaca mulatta) possesses multiple HLA-A-like (Mamu-A) genes, which parade varying degrees of polymorphism. The number and combination of transcribed Mamu-A genes present per chromosome display diversity in a population of Indian animals. At present, it is not clearly understood whether these different A region configurations are evolutionarily stable entities. To shed light on this issue, rhesus macaques from a Chinese population and a panel of cynomolgus monkeys (Macaca fascicularis) were screened for various A region-linked variations. Comparisons demonstrated that most A region configurations are old entities predating macaque speciation, whereas most allelic variation (>95%) is of more recent origin. The latter situation contrasts the observations of the major histocompatibility complex class II genes in rhesus and cynomolgus macaques, which share a high number of identical alleles (>30%) as defined by exon 2 sequencing.     

Comparative genetics of a highly divergent DRB microsatellite in different macaque species

The DRB region of the major histocompatibility complex (MHC) of cynomolgus and rhesus macaques is highly plastic, and extensive copy number variation together with allelic polymorphism makes it a challenging enterprise to design a typing protocol. All intact DRB genes in cynomolgus monkeys (Mafa) appear to possess a compound microsatellite, DRB-STR, in intron 2, which displays extensive length polymorphism. Therefore, this STR was studied in a large panel of animals, comprising pedigreed families as well. Sequencing analysis resulted in the detection of 60 Mafa-DRB exon 2 sequences that were unambiguously linked to the corresponding microsatellite. Its length is often allele specific and follows Mendelian segregation. In cynomolgus and rhesus macaques, the nucleotide composition of the DRB-STR is in concordance with the phylogeny of exon 2 sequences. As in humans and rhesus monkeys, this protocol detects specific combinations of different DRB-STR lengths that are unique for each haplotype. In the present panel, 22 Mafa-DRB region configurations could be defined, which exceeds the number detected in a comparable cohort of Indian rhesus macaques. The results suggest that, in cynomolgus monkeys, even more frequently than in rhesus macaques, new haplotypes are generated by recombination-like events. Although both macaque species are known to share several identical DRB exon 2 sequences, the lengths of the corresponding microsatellites often differ. Thus, this method allows not only fast and accurate DRB haplotyping but may also permit discrimination between highly related macaque species.     

Identification of MhcMafa-DRB Alleles in a Cohort of Cynomolgus Macaques of Vietnamese Origin

Cynomolgus macaques have been used widely to build a research model of infectious and chronic diseases, as well as in transplantation studies, where disease susceptibility and/or resistance are associated with the major histocompatibility complex (MHC). To better elucidate polymorphisms and genetic differences in the Mafa‐DRB locus, and facilitate the experimental use of cynomolgus macaques, we used pool screening combined with cloning and direct sequencing of polymerase chain reaction products to characterize MhcMafa‐DRB gene alleles in 153 Vietnamese cynomolgus macaques. We identified 30 Mafa‐DRB alleles belonging to 17 allelic lineages, including four novel sequences that had not been documented in earlier reports. The highest frequency allele was Mafa‐DRB*W27:04, which was present in 7 of 35 (20%) monkeys. The next most frequent alleles were Mafa‐DRB*3:07 and Mafa‐DRB*W7:01, which were detected in 5 of 35 (14.3%) and 4 of 35 (11.4%) of the monkeys, respectively. The high‐frequency alleles in this Vietnamese population may be high priority targets for additional characterization of immune functions. Only the DRB1*03 and DRB1*10 lineages were also present in humans, whereas the remaining alleles were monkey‐specific lineages. We found 25 variable sites by aligning the deduced amino acid sequences of 29 identified alleles. Evolutionary and population analyses based on these sequences showed that human, rhesus, and cynomolgus macaques share several Mhc‐DRB lineages and the shared polymorphisms in the DRB region may be attributable to the existence of interbreeding between rhesus and cynomolgus macaques. This information will promote the understanding of MHC diversity and polymorphism in cynomolgus macaques and increase the value of this species as a model for biomedical research. Am. J. Primatol. 74:958‐966, 2012. © 2012 Wiley Periodicals, Inc.     

Single‐Nucleotide Polymorphisms Reveal Patterns of Allele Sharing Across the Species Boundary Between Rhesus (Macaca mulatta) and Cynomolgus (M. fascicularis) Macaques

Both phenotypic and genetic evidence for asymmetric hybridization between rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques has been observed in the region of Indochina where both species are sympatric. The large‐scale sharing of major histocompatibility complex (MHC) class II alleles between the two species in this region supports the hypothesis that genes, and especially genes involved in immune response, are being transferred across the species boundary. This differential introgression has important implications for the incorporation of cynomolgus macaques of unknown geographic origin in biomedical research protocols. Our study found that for 2,808 single‐nucleotide polymorphism (SNP) markers, the minor allele frequencies (MAF) and observed heterozygosity calculated from a sample of Vietnamese cynomolgus macaques was significantly different from those calculated from samples of both Chinese rhesus and Indonesian cynomolgus macaques. SNP alleles from Chinese rhesus macaques were overrepresented in a sample of Vietnamese cynomolgus macaques relative to their Indonesian conspecifics and located in genes functionally related to the primary immune system. These results suggest that Indochinese cynomolgus macaques represent a genetically and immunologically distinct entity from Indonesian cynomolgus macaques. Am. J. Primatol. 75:135‐144, 2013. © 2012 Wiley Periodicals, Inc.     

Extensive sharing of MHC class II alleles between rhesus and cynomolgus macaques

In contrast to rhesus monkeys, substantial knowledge on cynomolgus monkey major histocompatibility complex (MHC) class II haplotypes is lacking. Therefore, 17 animals, including one pedigreed family, were thoroughly characterized for polymorphic Mhc class II region genes as well as their mitochondrial DNA (mtDNA) sequences. Different cynomolgus macaque populations appear to exhibit unique mtDNA profiles reflecting their geographic origin. Within the present panel, 10 Mafa-DPB1, 14 Mafa-DQA1, 12 Mafa-DQB1, and 35 Mafa-DRB exon 2 sequences were identified. All of these alleles cluster into lineages that were previously described for rhesus macaques. Moreover, about half of the Mafa-DPB1, Mafa-DQA1, and Mafa-DQB1 alleles and one third of the Mafa-DRB exon 2 sequences are identical to rhesus macaque orthologues. Such a high level of Mhc class II allele sharing has not been reported for primate species. Pedigree analysis allowed the characterization of nine distinct Mafa class II haplotypes, and seven additional ones could be deduced. Two of these haplotypes harbor a duplication of the Mafa-DQB1 locus. Despite extensive allele sharing, rhesus and cynomolgus monkeys do not appear to possess identical Mhc class II haplotypes, thus illustrating that new haplotypes were generated after speciation by recombination-like processes.     

MHC class I alleles influence set-point viral load and survival time in simian immunodeficiency virus-infected rhesus monkeys

In HIV-infected humans and SIV-infected rhesus macaques, host genes influence viral containment and hence the duration of the disease-free latency period. Our knowledge of the rhesus monkey immunogenetics, however, is limited. In this study, we describe partial cDNA sequences of five newly discovered rhesus macaque (Mamu) class I alleles and PCR-based typing techniques for the novel and previously published Mhc class I alleles. Using 15 primer pairs for PCR-based typing and DNA sequence analysis, we identified at least 21 Mhc class I alleles in a cohort of 91 SIV-infected macaques. The results confirm the presence of multiple class I genes in rhesus macaques. Of these alleles, Mamu-A*01 was significantly associated with lower set-point viral load and prolonged survival time. Mamu-A*1303 was associated with longer survival and a "novel" Mhc class I allele with lower set-point viral load. The alleles are frequent in rhesus macaques of Indian origin (12-22%). In addition, survival probability of individual SIV-infected rhesus monkeys increased with their number of alleles considered to be associated with longer survival. The results contribute to improve the interpretation and quality of preclinical studies in rhesus monkeys.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.