Mamu Class Research Articles

OR14: THE MACAQUE ALLELE MAMU-A1∗004 IS FUNCTIONALLY SIMILAR TO HLA-B∗57

Aim Rhesus macaques are a commonly used primate model for the study of viral infection. The class I MHC of these animals has been well studied at the genetic level. However functional ligandome characteristics such as peptide binding motif and length distribution have been elucidated in only a handful of macaque MHC (Mamu). Historically, Mamu ligandome characteristics have mimicked HLA-B despite the MAMU-A designation. Here we selected a high-frequency, uncharacterized, allele Mamu-A1∗004:01 to define the ligandome characteristics of this allele for comparison to HLA class I. Method Soluble MAMU-A1∗004:01 and HLA-B57:01 constructs were created and transfected these into the class I deficient cell line 721.221. sMHC harvested from transfected cells was affinity purified and ligand pools were isolated with an acid boil followed by 3 kDa cut-off ultrafiltration. Ligand pools were separated with two-dimentional LCMS. Data was acquired with a triple TOF mass spectrometer in information-dependant acquisition mode. Peptide sequences from the resulting MS2 fragment spectra were identified using the PEAKS and Mascot algorithms at a False Discovery Rate (FDR) of 1%. Results In this study, we identified 1447 ligands for Mamu-A1∗004. The peptide binding motif for MAMU-A1∗004 shows the P2 anchor as S, T, A and a PΩ anchor of W or F. This motif is strikingly similar to the HLA-B∗57:01 peptide binding motif. Indeed a comparison of the Mamu-A1∗004 and HLA-B∗57:01 eluted ligands from the same cell line shows that 32% of the Mamu-A1∗004 ligands are shared/identical with HLA-B∗57:01 ligands. Further, we established a Mamu class I peptide binding assay and demonstrated that known HLA-B57 HIV ligands bind to MAMU-A1∗004 with the same affinities. Although these allomorphs are similar in their motif, the length preferences are different: Compared to HLA-B57, Mamu-A1∗004 prefers nonamers and shows less tolerance for long (>13 amino acids) ligands. Conclusion Here we found that a common Rhesus macaque allele Mamu-A1∗004 is functionally homologous to HLA-B∗57:01. In humans, HLA-B∗57:01 is considered to be an important protective allele in HIV infections. These data enable the study of an HLA-B∗57:01-like molecule in non-human primate models of SIV infection. C. McMurtrey: Consultant; Company/Organization; Pure Protein LLC. R. Buchli: Employee; Company/Organization; Pure Protein LLC. W. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure Protein LLC.

Unique peptide-binding motif for Mamu-B*037:01: an MHC class I allele common to Indian and Chinese rhesus macaques

Indian and Chinese rhesus macaques are often used in biomedical research. Genetic analyses of the major histocompatibility class I region have revealed that these macaques display a substantial level of polymorphism at Mamu-A and Mamu-B loci, which have been subject to duplication. Only a few Mamu class I allotypes are characterised for their peptide-binding motifs, although more information of this nature would contribute to a better interpretation of T cell-mediated immune responses. Here, we present the results of the characterisation of the functional properties of Mamu-B*037:01, an allotype commonly encountered in rhesus macaques of Indian and Chinese origin. Mamu-B*037:01 is seen to have a strong preference for acidic amino acids at the third residue, and for arginine, lysine, and tyrosine at the carboxyl terminus. This peptide-binding motif is not described in the human population.

37-OR

Aim Cynomolgus macaque (Macaca fascicularis = Mafa) and Rhesus macaque (Macaca mulatta = Mamu) are widely used as alternative nonhuman primate models for studying HIV, transplantation, and vaccines since they are anatomically and physiologically similar to humans. In this study we characterize naturally loaded peptides from two Mafa (A ∗ 31 & B ∗ 49) and one Mamu (B ∗ 47) class I molecule. Methods An MHC Class I-deficient human B cell line was transfected with Mafa and Mamu soluble MHC (sMHC) DNA constructs and selected with Geneticin. Clones were tested for sMHC class I production by ELISA, and “best producers” were single cell sorted, expanded, and retested. Cell pharms were seeded and 25 mg of class I MHC harvested. Mafa and Mamu class I MHC were purified by affinity chromatography, peptide ligands eluted in acetic acid, and the pooled peptide ligands were subjected to 14 cycles of Edman degradation. Pooled peptides were HPLC fractionated and subjected to nanospray mass spectrometry (MS) in order to characterize individual peptide ligands. Results MS analysis data show that Mafa molecules present peptides similar to those presented by human HLA class I molecules. The peptide lengths and source are consistent with previous reports of class I MHC. The two Mafa molecules in this study have peptide-binding motifs characteristic of HLA-B molecules while Mamu peptide ligands are not consistent with previously reported MHC class I. Conclusions Mafa class I molecules exhibit HLA-B-like cross-species peptide presentation, while Mamu class I molecules do not show similarities with human HLA class I molecules. The characterization of endogenously loaded Mafa-B ∗ 49 MHC class I peptides shows that it is comparable to human HLA-B ∗ 4405, and Mafa-A ∗ 31 to human HLA-B ∗ 1509 and B ∗ 1510. These data suggest that the MHC typing of Non-Human Primates for comparable MHC should proceed the use of these animal models when establishing correlates of immunity.

Immunization with recombinant macaque major histocompatibility complex class I and II and human immunodeficiency virus gp140 inhibits simian–human immunodeficiency virus infection in macaques

Genetic, epidemiological and experimental evidence suggest that the major histocompatibility complex (MHC) is critical in controlling human immunodeficiency virus (HIV) infection. The objectives of this study were to determine whether novel recombinant Mamu MHC constructs would elicit protection against rectal challenge with heterologous simian-human immunodeficiency virus (SHIV) strain SF162.P4 in rhesus macaques. Mamu class I and II gene products were linked together with HIV gp140, simian immunodeficiency virus (SIV) p27 and heat-shock protein 70 to dextran. The vaccine was administered to two groups, each consisting of nine macaques, either subcutaneously (SC), or rectally and boosted by SC immunization. The controls were untreated or adjuvant-treated animals. Repetitive rectal challenges with up to ten doses of SHIV SF162.P4 showed a significant decrease in the peak and sequential viral RNA concentrations, and three macaques remained uninfected, in the nine SC-immunized animals, compared with infection in all nine controls. Macaques immunized rectally followed by SC boosters showed a less significant decrease in both sequential and peak viral loads compared with the SC-immunized animals, and all were infected following rectal challenge with SHIV SF162.P4. Plasma and mucosal IgG and IgA antibodies to Mamu class I alleles and HIV gp120, as well as to RANTES (regulated upon activation, normal T-cell expressed, and secreted; CCR5) were increased, and showed significant inverse correlations with the peak viral load. These results suggested that allo-immunization with recombinant MHC constructs linked to HIV-SIV antigens merits further investigation in preventing HIV-1 infection.

The role of MHC class I allele Mamu-A*07 during SIV(mac)239 infection.

Virus-specific CD8(+) T cells play an important role in controlling HIV/SIV replication. These T cells recognize intracellular pathogen-derived peptides displayed on the cell surface by individual MHC class I molecules. In the SIV-infected rhesus macaque model, five Mamu class I alleles have been thoroughly characterized with regard to peptide binding, and a sixth was shown to be uninvolved. In this study, we describe the peptide binding of Mamu-A1*007:01 (formerly Mamu-A*07), an allele present in roughly 5.08% of Indian-origin rhesus macaques (n = 63 of 1,240). We determined a preliminary binding motif by eluting and sequencing endogenously bound ligands. Subsequently, we used a positional scanning combinatorial library and panels of single amino acid substitution analogs to further characterize peptide binding of this allele and derive a quantitative motif. Using this motif, we selected and tested 200 peptides derived from SIV(mac)239 for their capacity to bind Mamu-A1*007:01; 33 were found to bind with an affinity of 500nM or better. We then used PBMC from SIV-infected or vaccinated but uninfected, A1*007:01-positive rhesus macaques in IFN-γ Elispot assays to screen the peptides for T-cell reactivity. In all, 11 of the peptides elicited IFN-γ(+) T-cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultradeep pyrosequencing demonstrated the accumulation of amino acid substitutions within four of these six regions, suggestive of selective pressure on the virus by antigen-specific CD8(+) T cells. Thus, it appears that Mamu-A1*007:01 presents SIV-derived peptides to antigen-specific CD8(+) T cells and is part of the immune response to SIV(mac)239.

Erratum to: Rhesus macaque MHC class I molecules show differential subcellular localizations

The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding α1 and α2 domains, suggesting failure of peptide binding is responsible for retaining ‘intracellular’ Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes.

Rhesus macaque MHC class I molecules show differential subcellular localizations

The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding α1 and α2 domains, suggesting failure of peptide binding is responsible for retaining ‘intracellular’ Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes.

Identification of a Mamu Class I-restricted listeriolysin T-cell epitope in rhesus macaques infected with Listeria monocytogenes (LM) (129.13)

Abstract LM is a gram positive foodborne pathogen that causes a flu-like illness in immunocompetent patients, but immunocompromised patients can develop septicemia and meningitis resulting in a high mortality rate. The primate model of LM infection is being used to investigate both innate and adaptive components of immunity in an animal model that is more relevant to human disease. Cytotoxic T lymphocytes were isolated from rhesus monkeys infected with LM by nasogastric intubation and used to identify Class I-restricted LM epitopes. Peptides were eluted from the surface of syngeneic B lymphocytes that were incubated overnight with heat-killed LM, and then peptides were separated by reversed-phase HPLC. An 11-mer peptide was identified that induced significant target cell lysis. The amino acid sequence of this peptide was determined by Edman degradation and confirmed by mass spectrometry. The sequence of this peptide encompassed amino acids 220-230 of the listeriolysin protein. Synthetic peptides were synthesized according to this sequence. The CTL responses induced by these peptides were comparable to the eluted native peptide.

Novel simian immunodeficiency virus CTL epitopes restricted by MHC class I molecule Mamu-B*01 are highly conserved for long term in DNA/MVA-vaccinated, SHIV-challenged rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques provides an excellent model for investigating the basis of protective immunity against human immunodeficiency virus (HIV). One limitation of this model, however, has been the availability of a small number of known MHC class I-restricted CTL epitopes for investigating virus-specific immune responses. We assessed CTL responses against SIV Gag in a cohort of DNA/modified vaccinia virus Ankara (MVA)-vaccinated/simian-human immunodeficiency virus (SHIV)-challenged rhesus macaques. Here, we report the identification of five novel SIV CTL epitopes in Gag for the first time (Gag(39-46) NELDRFGL, Gag(169-177) EVVPGFQAL, Gag(198-206) AAMQIIRDI, Gag(257-265) IPVGNIYRR and Gag(296-305) SYVDRFYKSL) that are restricted by the common MHC class I molecule Mamu-B*01. CTL responses to these epitopes were readily detected in cryopreserved PBMC in multiple animals up to 62 weeks post-infection, both by IFN-gamma enzyme-linked immunospot assay and intracellular IFN-gamma staining. Importantly, viral sequencing results revealed that these epitopes are highly conserved in the SIV-challenged macaques over a long period of time, indicating functional constraints in these regions. Moreover, the presence of CTL responses targeting these epitopes has been confirmed in two independent cohorts of rhesus macaques that have been challenged by SHIV or SIV. Our findings provide valuable candidates for poly-epitope vaccines and for long-term quantitative monitoring of epitope-specific CD8(+) responses in the context of this common Mamu class I allele. It may thus help increase the supply of rhesus macaques in which epitope-specific immunity can be studied in the context of SIV vaccine design.

Evolutionary stability of MHC class II haplotypes in diverse rhesus macaque populations.

A thoroughly characterized breeding colony of 172 pedigreed rhesus macaques was used to analyze exon 2 of the polymorphic Mamu- DPB1, -DQA1, -DQB1, and - DRB loci. Most of the monkeys or their ancestors originated in India, though the panel also included animals from Burma and China, as well as some of unknown origin and mixed breeds. In these animals, mtDNA appears to correlate with the aforementioned geographic origin, and a large number of Mamu class II alleles were observed. The different Mamu- DPB1 alleles were largely shared between monkeys of different origin, whereas in humans particular alleles appear to be unique for ethnic populations. In contrast to Mamu-DPB1, the highly polymorphic - DQA1/DQB1 alleles form tightly linked pairs that appear to be about two-thirds population specific. For most of the DQA1/DQB1 pairs, Mamu- DRB region configurations present on the same chromosome have been ascertained, resulting in 41 different -DQ/DRB haplotypes. These distinct DQ/DRB haplotypes seem to be specific for monkeys of a determined origin. Thus, in evolutionary terms, the Mamu-DP, -DQ, and -DR regions show increasing instability with regard to allelic polymorphism, such as for -DP/DQ, or gene content and allelic polymorphism, such as for -DR, resulting in population-specific class II haplotypes. Furthermore, novel haplotypes are generated by recombination-like events. The results imply that mtDNA analysis in combination with Mhc typing is a helpful tool for selecting animals for biomedical experiments.

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.