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D I Watkins

Publications and source records attributed to D I Watkins.

At least 55 records · Page 3Linked to original sources
PubMed1999

Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef.

Cytotoxic T-lymphocyte (CTL) responses to human immunodeficiency virus arise early after infection, but ultimately fail to prevent progression to AIDS. Human immunodeficiency virus may evade the CTL response by accumulating amino-acid replacements within CTL epitopes. We studied 10 CTL epitopes during the course of simian immunodeficiency virus disease progression in three related macaques. All 10 of these CTL epitopes accumulated amino-acid replacements and showed evidence of positive selection by the time the macaques died. Many of the amino-acid replacements in these epitopes reduced or eliminated major histocompatibility complex class I binding and/or CTL recognition. These findings strongly support the CTL 'escape' hypothesis.

Amino Acid Sequence↗
PubMed1999

Major histocompatibility complex class I genes in primates: co-evolution with pathogens.

The major histocompatibility complex (MHC) is the most polymorphic genetic system known, playing a central role in the cellular immune response to pathogens. The relationship between the MHC of humans and non-human primates has increased our understanding of MHC evolution and how polymorphism of this gene family may have been generated. We will review MHC class I evolution in great apes and Old World and New World primates and discuss new data from the simian immunodeficiency virus/rhesus monkey animal model that demonstrate the role of MHC class I alleles in selecting for new populations of viruses. This suggests that certain pathogens co-evolve with the MHC class I molecules they encounter in a population.

Amino Acid Sequence↗
PubMed1999

The simian immunodeficiency virus envelope glycoprotein contains two epitopes presented by the Mamu-A*01 class I molecule.

Cytotoxic T lymphocyte (CTL) responses against the simian immunodeficiency virus (SIV) envelope and Gag proteins were monitored in a Mamu-A*01-positive rhesus macaque infected with SIVsmE660. Peripheral blood mononuclear cells (PBMC) cultured with synthetic peptides spanning the entire gp160 and Gag coding region recognized a total of three epitopes. One located in Gag was identified as the previously described Mamu-A*01-restricted p11cC-->M epitope (CTPYDINQM). The other two epitopes, designated p15m and p54m, were located in the gp160 envelope protein. Both were nine amino acids in length and were predicted to bind Mamu-A*01 because they contained proline and leucine residues at positions 3 and 9, respectively. Indeed, expression of this class I major histocompatibility complex molecule was required for target cell recognition by envelope-specific CD8(+) T cells directed against both epitopes. These Mamu-A*01-restricted epitopes in the SIV envelope will be useful for monitoring immune responses in vaccinated or infected animals.

Amino Acid Sequence↗
PubMed1999

Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen.

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed.

Animals↗
PubMed1998

Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus.

The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.

Animals↗
PubMed1998

Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex.

A tetrameric recombinant major histocompatibility complex (MHC) class I-peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and beta2-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C-M) representing the optimal nine-amino acid peptide of Mamu-A*01-restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C-M complex. Tetrameric Mamu-A*01/p11C, C-M complex bound to T cells of SIVmac-infected, Mamu-A*01(+), but not uninfected, Mamu-A*01(+), or infected, Mamu-A*01(-) rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01(+) rhesus monkeys was only found in the cluster of differentiation (CD)8alpha/beta+ T lymphocyte subset and the percentage of CD8alpha/beta+ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01+ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C-M complex-binding, but not nonbinding, CD8alpha/beta+ T cells. Furthermore, the percentage of CD8alpha/beta+ T cells binding this tetrameric Mamu-A*01/p11C, C-M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.

Animals↗
PubMed1998

The MHC-E locus is the most well conserved of all known primate class I histocompatibility genes.

The HLA-E locus is characterized by limited polymorphism and low levels of cell surface expression. However, the function of the products of this nonclassical MHC class I gene remains unknown. To evaluate the conservation of the MHC-E locus throughout anthropoid primate evolution, we identified the homologue of the HLA-E locus in six different New World monkey species. Full-length sequencing of MHC-EcDNAs in four unrelated cotton-top tamarins (Saguinus oedipus) revealed no evidence for polymorphism. Using the PCR, denaturing gradient gel electrophoresis, and direct sequencing, we also identified MHC-E alleles in five other New World monkey species, representing all extant platyrrhine families. In contrast to all other classical and nonclassical MHC class I genes in primates, the rate of synonymous nucleotide substitution is much greater than the rate of nonsynonymous nucleotide substitution within exons 2 and 3 encoding the peptide binding region (PBR) in MHC-E genes. The PBR of the MHC-E molecule, therefore, has evolved under purifying selective pressures, and the very unusual evolutionary history of this ancient gene provides further evidence that the products of the HLA-E locus serve a critical immunological function. Given the remarkable conservation of the PBR during primate evolution, this critical immunological function is probably related to the peptide binding ability of the MHC-E protein.

Amino Acid Sequence↗
PubMed1998

Two different primate species express an identical functional MHC class I allele.

The products of the highly polymorphic and variable major histocompatibility complex (MHC) class I loci play a crucial role in host defenses against infectious disease. While similar alleles have been found in closely related species, sharing of a functional MHC class I allele between two species has never been reported. Here we show that an identical functional MHC class I molecule is present in two different primate species with an approximate divergence time of 0.7 million years. Lymphocytes from the red-crested tamarin (Saguinus geoffroyi) expressed an MHC class I allele (Sage-G*01) that was identical in coding sequence to an MHC class I allele (Saoe-G*08) found in the cotton-top tamarin (Saguinus oedipus). Furthermore, influenza virus-specific cytotoxic T lymphocytes (CTLs) generated in the cotton-top tamarin killed lymphocytes expressing the influenza virus nucleoprotein (NP) from the red-crested tamarin. Since the influenza virus NP epitope is bound by Saoe-G*08 in the cotton-top tamarin, it is likely that this molecule is functional in both species. These data provide the first evidence that functional MHC class I molecules can be maintained entirely intact in two separate species.

Alleles↗
PubMed1998

Denaturing gradient gel electrophoresis: a rapid method for differentiating BoLA-DRB3 alleles.

The products of the BoLA-DRB3 locus are important molecules in the bovine immune response. Several techniques have been used to study and define this locus but they are generally time consuming and limited in their ability to detect novel alleles. In this study we used denaturing gradient gel electrophoresis (DGGE), and direct sequencing, for BoLA-DRB3-typing. First, modified locus-specific primers were used in polymerase chain reaction (PCR) to amplify a 240 bp fragment of exon 2 of BoLA-DRB3 from the genomic DNA of 22 cattle and one pair of twin calves. The reverse primer included a GC-rich clamp to improve the physical separation of the BoLA-DRB3 alleles by DGGE. The denaturing gradient needed to produce separation of alleles was determined using perpendicular DGGE, and this gradient was then applied to parallel denaturing gels. The optimal time for producing allele separation was determined using a time-series analysis. The bands representing individual BoLA-DRB3 alleles were excised from the gels, reamplified, and the nucleotide sequence determined using fluorescent-based automated cycle sequencing. The nucleotide sequences of the separated bands were then compared to published BoLA-DRB3 alleles. A gradient of 10-15% acrylamide combined with a 15-50% ureaformamide gradient was successfully used to separate BoLA-DRB3 alleles in all individuals examined. Nucleotide sequencing showed that the 24 animals possessed 13 BoLA-DRB3 alleles, all of which have been previously described. The BoLA-DRB3 genotypes included 20 heterozygotes and two homozygotes. Three BoLA-DRB3 alleles were seen in each of the twin calves, possibly due to leukochimerism. The technique is reliable and rapid, and avoids cloning alleles prior to nucleotide sequencing and therefore offers distinct advantages over previous techniques for BoLA-DRB3-typing.

Alleles↗
PubMed1998

The rhesus monkey analogue of human lymphocyte antigen-G is expressed primarily in villous syncytiotrophoblasts.

The human placenta expresses the nonclassical major histo-compatibility complex (MHC) class I molecule, human lymphocyte antigen (HLA)-G, which may contribute to the establishment of maternal-fetal immune tolerance. Although the HLA-G ortholog of the rhesus monkey, Mamu-G, is a pseudogene, another nonclassical MHC class I locus, Mamu-AG, is expressed in the rhesus monkey placenta. Mamu-AG encodes MHC class I A locus-related molecules that exhibit all the characteristics of human HLA-G, including limited polymorphism and a truncated cytoplasmic domain. We have examined MHC class I glycoprotein and Mamu-AG mRNA expression in the rhesus placenta and in cultured trophoblasts. Immunocytochemical analysis of rhesus placental tissues with the W6/32 monoclonal antibody demonstrated a high level of MHC class I expression in villous syncytiotrophoblasts, whereas villous cytotrophoblasts were largely MHC class I negative. Only low levels of MHC class I expression were seen in extravillous cytotrophoblasts of cell columns and the trophoblastic shell. In situ hybridization demonstrated that Mamu-AG mRNAs were expressed at a high level in first-trimester villous syncytiotrophoblasts. MHC class I and Mamu-AG expression was significantly up-regulated during in vitro culture and differentiation of freshly isolated villous cytotrophoblasts into syncytiotrophoblasts. Preferential Mamu-AG expression in syncytiotrophoblasts suggests that rhesus monkey MHC class I-bearing trophoblasts could potentially interact with maternal peripheral blood lymphocytes rather than with uterine decidual lymphocytes as has been proposed for human trophoblasts.

Animals↗
PubMed1997

Evolutionary instability of the major histocompatibility complex class I loci in New World primates.

Homologues of the human major histocompatibility complex (MHC) HLA-A, -B, -E, -F, and -G loci are present in all the Catarrhini (Old World primates, apes, and humans), and some of their allelic lineages have survived several speciation events. Analysis of 26 MHC class I cDNAs from seven different genera of New World primates revealed that the Callitrichinae (tamarins and marmosets) are an exception to these rules of MHC stability. In gene trees of primate MHC class I genes, sequences from the Callitrichinae cluster in a genus-specific fashion, whereas in the other genera of New World primates, as in the Catarrhini, they cluster in a transgeneric way. The genus-specific clustering of the Callitrichinae cDNAs indicates that there is no orthology between MHC class I loci in genera of this phyletic group. Additionally, the Callitrichinae genera exhibit limited variability of their MHC class I genes, in contrast to the high variability displayed by all other primates. Each Callitrichinae genus, therefore, expresses its own set of MHC class I genes, suggesting that an unusually high rate of turnover of loci occurs in this subfamily. The limited variability of MHC class I genes in the Callitrichinae is likely the result of the recent origin of these loci.

Animals↗
PubMed1997

Identification of a novel MHC class I gene, Mamu-AG, expressed in the placenta of a primate with an inactivated G locus.

Maternal tolerance of the fetal allograft remains poorly understood. In humans, expression of the highly polymorphic classical HLA-A and HLA-B loci is suppressed, while expression of the nonclassical HLA-G locus is up-regulated at the maternal-fetal interface. Like other nonclassical MHC class I molecules, HLA-G exhibits limited diversity, but certain characteristics of HLA-G distinguish it from other nonclassical MHC class I molecules: it has a truncated cytoplasmic domain, it is the product of alternatively spliced mRNAs, and it is expressed primarily in the placenta. We have examined MHC class I expression in the placenta of the rhesus monkey to determine whether this animal is a suitable model in which to study the function of HLA-G. Although the rhesus monkey possesses orthologs of many MHC class I and II loci found in humans, the HLA-G ortholog is a pseudogene in this nonhuman primate species. In this study, we report the identification of a novel nonclassical MHC class I locus expressed in the placenta of the rhesus monkey, Mamu-AG (Macaca mulatta-AG). Although unrelated to HLA-G, Mamu-AG encodes glycoproteins with all of the characteristics of HLA-G. These Mamu-AG glycoproteins are limited in their diversity, possess truncated cytoplasmic domains, are the products of alternatively spliced mRNAs, and their expression is restricted to the placenta. Taken together, these data suggest that convergent evolution may have resulted in the expression of a unique nonclassical MHC class I molecule in the rhesus monkey placenta, and that the common structural features of Mamu-AG and HLA-G may be functionally significant.

Alleles↗
PubMed1997

Immunodominance of a single CTL epitope in a primate species with limited MHC class I polymorphism.

MHC class I molecules play a crucial role in immunity to viral infections by presenting viral peptides to cytotoxic T lymphocytes. One of the hallmarks of MHC class I genes in outbred populations is their extraordinary polymorphism, yet the significance of this diversity is poorly understood. Certain species with reduced MHC class I diversity, such as the cotton-top tamarin (Saguinus oedipus), are more susceptible to fatal viral infections. To explore the relationship between this primate's limited MHC class I diversity and its susceptibility to viruses, we infected five cotton-top tamarins with influenza virus. Every tamarin recognized the same immunodominant CTL epitope of the influenza nucleoprotein. Surprisingly, this nucleoprotein peptide was bound by Saoe-G*08, an MHC class I molecule expressed by every cotton-top tamarin. Two tamarins also made a subdominant response to an epitope of the matrix (M1) protein. This peptide appeared to be bound by another common MHC class I molecule. With the exception of an additional subdominant response to the polymerase (PB2) protein in one individual, no other influenza-specific CTL responses were detected. In populations or species with limited MHC class I polymorphism like the cotton-top tamarin, a dependence on shared MHC class I molecules may enhance susceptibility to viral infection, since viruses that evade MHC class I-restricted recognition in one individual will likely evade recognition in the majority of individuals.

Animals↗
PubMed1997

Split tolerance induced by immunotoxin in a rhesus kidney allograft model.

BACKGROUND: Renal allografts were performed in rhesus monkeys using FN18-CRM9, a potent immunotoxin capable of depleting T cells to less than 1% of baseline levels in blood and lymph nodes, as a preparative agent. We have recently reported that animals pretreated with FN18-CRM9 1 week before transplantation without further immunosuppression had prolonged graft survival time compared with control animals, and frequently became tolerant. METHODS: This report examines the alloimmune responses of recipient monkeys to the donor, including cytotoxic T lymphocyte precursor (CTLp) frequency, mixed lymphocyte response, and antidonor IgG response. RESULTS: CTLp frequencies declined significantly (P<0.01) after FN18-CRM9 treatment and renal transplantation. This decline in CTLp was initially nonspecific, as CTLp frequencies against third-party animals also declined (P<0.01). The decrease in CTLp was maintained in five of five animals tested 6 months after transplant. However, unresponsiveness was limited to the CTL arm of the immune response as antidonor IgG was detected in four of four animals tested, and the 5-day mixed lymphocyte response stimulation index and relative response were not significantly different before and after transplant. In long-term survivors (>150 days), an increase in anti-third-party CTLp was detected 1 month after grafting with third-party skin. No change was seen in the antidonor CTLp frequency after donor skin grafting, indicating that a specific defect in the antidonor CTL response had developed. CONCLUSIONS: These data suggest that FN18-CRM9 treatment of rhesus monkeys allows the development of specific down-regulation of antidonor CTL activity in renal allograft recipients.

Animals↗
PubMed1997

Identification of new mamu-DRB alleles using DGGE and direct sequencing.

Rhesus macaques represent important animal models for biomedical research. The ability to identify macaque major histocompatibility complex (Mhc) alleles is crucial for fully understanding these models of autoimmune and infectious disease. Here we describe a rapid and unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. Importantly, our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different Mhc-DRB lineages. Interestingly, two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. This new technique should also contribute to ongoing studies of Mhc function and evolution in many different species of nonhuman primates.

Alleles↗
PubMed1997

Identification of a new HLA-B*08 variant, HLA-B*0804.

The HLA-B locus is the most polymorphic locus known with currently over 100 different alleles described. Many of these alleles encode variants of the serologically-defined tissue transplantation antigens. This high level of diversity makes accurate tissue typing difficult. Here we present the sequence of a new HLA-B*08 variant, HLA-B*0804, found in Caucasian siblings JH and PF serologically typed as HLA-B51/B59 and HLA-B59/B60, respectively. Additionally, DNA-based typing by the polymerase chain reaction using sequence-specific primers (PCR-SSP) identified HLA-B*51 in JH and HLA-B*4001 in PF. However, PCR-SSP failed to identify a second allele in either of these individuals. The unusual finding of a B59 antigen in a Caucasian and the discrepant molecular typing results suggested that these individuals might express novel HLA molecules. Using denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we characterized a novel HLA-B*08 variant, HLA-B*0804. The presence of this allele was confirmed by cloning and sequencing. HLA-B*0804 differed from HLA-B*0801 by only one nucleotide substitution resulting in an amino acid replacement of phenylalanine by serine at position 67. Incidentally, this single nucleotide difference was sufficient to prevent amplification by PCR-SSP. This striking difference between both the serologically typed antigen and the PCR-SSP-identified allele compared to the sequenced allele supports the use of sequence-based typing for the analysis of HLA class I locus alleles.

Amino Acid Sequence↗
PubMed1997

HLA-B typing by allele separation followed by direct sequencing.

Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous molecular typing method for the alleles at this locus. Here we describe a technique for the direct sequencing of HLA-B alleles. Initially, HLA-B alleles were PCR-amplified after locus-specific reverse transcription of RNA. Alleles were then separated using denaturing gradient gel electrophoresis (DGGE), which separates DNA fragments based on their sequence composition. Amplification products were excised from the gel and eluted DNA was reamplified and directly sequenced. The derived sequences were aligned to a database of published HLA-B sequences, and an initial allele assignment was made. This approach was theoretically sufficient to type 92 of the 118 known HLA-B alleles. The majority of the remaining 26 alleles contain differences at the beginning of exon 2, a region outside the DGGE-separated PCR products. Therefore, we used heterozygous sequencing of this region to identify 19 of these 26 alleles, raising the resolution power to 111 alleles. Using this technique, we analyzed immortalized cell lines and blood samples from several different sources. Nine immortalized cell lines were obtained from the 10th International Histocompatibility Workshop (IHWS) and nine were derived from aboriginal peoples. Additionally, 25 blood samples were acquired from a panel of donors previously shown to be difficult to type using serological techniques. Altogether, using this new method of allele separation by DGGE followed by direct sequencing, we typed 52 different alleles from 57 individuals, covering 40 serological specificities.

Alleles↗