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David I Watkins

Publications and source records attributed to David I Watkins.

At least 19 recordsLinked to original sources
PubMed2006

The antiviral efficacy of simian immunodeficiency virus-specific CD8+ T cells is unrelated to epitope specificity and is abrogated by viral escape.

CD8(+) T lymphocytes appear to play a role in controlling human immunodeficiency virus (HIV) replication, yet routine immunological assays do not measure the antiviral efficacy of these cells. Furthermore, it has been suggested that CD8+ T cells that recognize epitopes derived from proteins expressed early in the viral replication cycle can be highly efficient. We used a functional in vitro assay to assess the abilities of different epitope-specific CD8+ T-cell lines to control simian immunodeficiency virus (SIV) replication. We compared the antiviral efficacies of 26 epitope-specific CD8+ T-cell lines directed against seven SIV epitopes in Tat, Nef, Gag, Env, and Vif that were restricted by either Mamu-A*01 or Mamu-A*02. Suppression of SIV replication varied depending on the epitope specificities of the CD8+ T cells and was unrelated to whether the targeted epitope was derived from an early or late viral protein. Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines were consistently superior at suppressing viral replication compared to the other five SIV-specific CD8+ T-cell lines. We also investigated the impact of viral escape on antiviral efficacy by determining if Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines could suppress the replication of an escaped virus. Viral escape abrogated the abilities of Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T cells to control viral replication. However, gamma interferon (IFN-gamma) enzyme-linked immunospot and IFN-gamma/tumor necrosis factor alpha intracellular-cytokine-staining assays detected cross-reactive immune responses against the Gag escape variant. Understanding antiviral efficacy and epitope variability, therefore, will be important in selecting candidate epitopes for an HIV vaccine.

Animals↗
PubMed2006

Not all cytokine-producing CD8+ T cells suppress simian immunodeficiency virus replication.

Current assays of CD8+ T-lymphocyte function measure cytokine production rather than the ability of these lymphocytes to suppress viral replication. Here we show that CD8+ T-cell clones recognizing the same epitope vary enormously in the ability to suppress simian immunodeficiency virus SIVmac239 replication in an in vitro suppression assay. However, all Nef(165-173)IW9- and Vif(66-73)HW8-specific clones from elite controllers effectively suppressed SIV replication. Interestingly, in vitro suppression efficacy was not always associated with the ability to produce gamma interferon, tumor necrosis factor alpha, or interleukin-2.

Animals↗
PubMed2006

Polymorphisms in eight host genes associated with control of HIV replication do not mediate elite control of viral replication in SIV-infected Indian rhesus macaques.

Polymorphisms in several host genes in HIV-infected individuals facilitate slow progression to AIDS. We have identified several SIV-infected Indian rhesus macaques that naturally control viral replication. We investigated whether spontaneous control of SIV in any of these animals could be explained by mutations in host genes. Such variables could confound studies of associations between MHC class I alleles and control of viral replication. We searched for polymorphisms in CCR5, CXCR6, GPR15, RANTES, IL-10, APOBEC3G, TNF-alpha, and TSG101 and looked for associations with decreased viral replication. We did not detect any correlations between plasma viral concentration and polymorphisms in host genes examined in this study. In addition, we did not find the polymorphisms present in humans in any of our macaques.

Animals↗
PubMed2006

Premature induction of an immunosuppressive regulatory T cell response during acute simian immunodeficiency virus infection.

Here we report the results of an investigation into the possibility that one mechanism responsible for the establishment of persistent human immunodeficiency virus infection is an early regulatory T (Treg) cell response that blunts virus-specific responses. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model, we show that, indeed, viral replication and immune activation in lymphatic tissue drive a premature immunosuppressive response, with dramatic increases in the frequencies of CD4+CD25+FOXP3+ Treg cells, transforming growth factor- beta 1+ cells, interleukin-10+ cells, and indoleamine 2,3-dioxygenase+CD3+ cells. When we compared SIV infection with rhesus cytomegalovirus (RhCMV) infection, we found that the frequency of Treg cells paralleled the magnitude of immune activation during both infections but that the magnitude of immune activation and of the Treg cell response were lower and peaked much later during RhCMV infection. Importantly, the frequency of Treg cells inversely correlated with the magnitude of the SIV-specific cytotoxic T lymphocyte response. We conclude that an early Treg cell response during acute SIV infection may contribute to viral persistence by prematurely limiting the antiviral immune response before infection is cleared.

Animals↗
PubMed2006

DNA immunization in combination with effective antiretroviral drug therapy controls viral rebound and prevents simian AIDS after treatment is discontinued.

DNA immunization in conjunction with antiretroviral therapy was evaluated in SIV-infected rhesus macaques treated with [R]-9-[2-phosphonylmethoxypropyl]adenine (PMPA). Macaques were immunized monthly with DNA vaccines expressing either SIV gag/tat or SIV gag/tat and 19 CD8+ T cell epitopes during 7 months of therapy. Half the animals from each group were additionally immunized before infection. Only 60% of the animals (4 controls, 20 vaccinated) responded to PMPA (ART responders). All 4 ART responder controls demonstrated viral rebound or CD4 decline after PMPA was withdrawn. In contrast, 17 of 20 vaccinated ART responders contained viral rebound for over 7 months after PMPA was withdrawn. Viral control correlated with stable CD4 counts, higher lymphoproliferation and an increase in the magnitude and breadth of the CD8+ T cell response. Immunizing before infection or with multi-epitopes enhanced these effects. These results demonstrate that DNA immunization during antiretroviral therapy may be an effective strategy to treat HIV infection.

Adenine↗
PubMed2006

HIV vaccine design: insights from live attenuated SIV vaccines.

The International AIDS Vaccine Initiative has established a consortium to elucidate mechanisms of protection conferred by live attenuated simian immunodeficiency virus vaccines in monkeys. Here, the strategies defining key components of the protective immune response elicited by these vaccines are discussed.

AIDS Vaccines↗
PubMed2006

Initiation of antiretroviral therapy during chronic SIV infection leads to rapid reduction in viral loads and the level of T-cell immune response.

In the present era of increasing resistance of human immunodeficiency virus (HIV) to antiviral drugs, exploration of adjunct therapies directed at immune responses in combination with antiretroviral drugs may be of value for the treatment of acquired immunodeficiency syndrome. In this study, we designed a model for immune therapy using SIVmac251 infection in rhesus macaques. We explored the outcomes of primary infection on viral loads and the resulting T-cell immune responses in primates. The SIV-infected rhesus macaque model exhibited features similar to those observed in HIV-1 infection of humans. Major histocompatibility complex (MHC) segregation with viral loads were found to associate with viral containment and hence the duration of the disease-free latency period. Thus a better understanding of the relative roles of MHC class I allele in control of viral replication may provide important information for prophylactic or therapeutic vaccine designs. Mamu-A01 is significantly associated with higher immune response and control of viral replication. This allele is frequent in rhesus macaques of Indian origin (22%). Interestingly, Mamu-B01 (26% animals) was associated with lower immune responses and higher viral loads. Another allele, A08 was also predominantly present in 37% of the animals in this study. We observed higher viral replication in individual SIV-infected rhesus monkeys that did not demonstrate strong cellular immune responses. The results are important for understanding SIV disease progression in different MHC Mamu alleles and also for improving the interpretation and quality of pre-clinical studies in rhesus monkeys.

Alleles↗
PubMed2006

Vaccine-induced cellular immune responses reduce plasma viral concentrations after repeated low-dose challenge with pathogenic simian immunodeficiency virus SIVmac239.

The goal of an AIDS vaccine regimen designed to induce cellular immune responses should be to reduce the viral set point and preserve memory CD4 lymphocytes. Here we investigated whether vaccine-induced cellular immunity in the absence of any Env-specific antibodies can control viral replication following multiple low-dose challenges with the highly pathogenic SIVmac239 isolate. Eight Mamu-A*01-positive Indian rhesus macaques were vaccinated with simian immunodeficiency virus (SIV) gag, tat, rev, and nef using a DNA prime-adenovirus boost strategy. Peak viremia (P = 0.007) and the chronic phase set point (P = 0.0192) were significantly decreased in the vaccinated cohort, out to 1 year postinfection. Loss of CD4(+) memory populations was also ameliorated in vaccinated animals. Interestingly, only one of the eight vaccinees developed Env-specific neutralizing antibodies after infection. The control observed was significantly improved over that observed in animals vaccinated with SIV gag only. Vaccine-induced cellular immune responses can, therefore, exert a measure of control over replication of the AIDS virus in the complete absence of neutralizing antibody and give us hope that a vaccine designed to induce cellular immune responses might control viral replication.

Animals↗
PubMed2006

The high-frequency major histocompatibility complex class I allele Mamu-B*17 is associated with control of simian immunodeficiency virus SIVmac239 replication.

Particular HLA alleles are associated with reduced human immunodeficiency virus replication. It has been difficult, however, to characterize the immune correlates of viral control. An analysis of the influence of major histocompatibility complex class I alleles on viral control in 181 simian immunodeficiency virus SIVmac239-infected rhesus macaques revealed that Mamu-B(*)17 was associated with a 26-fold reduction in plasma virus concentrations (P<0.001). Mamu-B(*)17 was also enriched in a group of animals that controlled viral replication by 1,000-fold [corrected] Even after accounting for this group, Mamu-B(*)17 was associated with an eightfold reduction in plasma virus concentrations (P<0.001). Mamu-B(*)17-positive macaques could, therefore, facilitate our understanding of the correlates of viral control.

Alleles↗
PubMed2005

Within-host evolution of CD8+-TL epitopes encoded by overlapping and non-overlapping reading frames of simian immunodeficiency virus.

In order to understand the impact of overlapping reading frames on natural selection by host CD8+ T lymphocytes (CD8(+)-TL), we analyzed the pattern of nucleotide substitution in simian immunodeficiency virus (SIV) genomes sampled from populations at time of death in 35 rhesus monkeys. Both the mean number of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and the mean number of synonymous nucleotide substitutions per synonymous site (d(S)) were elevated in overlap regions in comparison to non-overlap regions. Mean d(N) exceeded mean d(S) in CD8(+)-TL epitopes restricted by the host's class I major histocompatibility complex molecules. This pattern, which is indicative of positive Darwinian selection favoring amino acid changes in these epitopes, was seen in both overlap and non-overlap regions; but mean d(N) was particularly elevated in restricted CD8(+)-TL epitopes encoded in overlap regions. Amino acid changes from the inoculum were defined as parallel if the same amino acid change occurred at the same site independently in two or more monkeys, and a surprisingly high proportion (71.9%) of observed amino acid changes throughout the SIV genome occurred in parallel in different monkeys. The proportion of parallel changes in restricted epitopes encoded by overlapping reading frames was still higher (80%), supporting the hypothesis that the interaction of positive selection and overlapping reading frames enhances the probability of convergent or parallel amino acid change.

Animals↗
PubMed2005

The high frequency Indian rhesus macaque MHC class I molecule, Mamu-B*01, does not appear to be involved in CD8+ T lymphocyte responses to SIVmac239.

Although the SIV-infected Indian rhesus macaque (Macaca mulatta) is the animal model most widely used for studying HIV infection, our current understanding of the functional macaque MHC class I molecules is limited. To date, SIV-derived CD8+ T lymphocyte epitopes from only three high frequency macaque MHC class I molecules have been extensively characterized. In this study, we defined the peptide-binding properties of the high frequency Indian rhesus macaque class I molecule, Mamu-B*01 ( approximately 26%). We first identified a preliminary binding motif by eluting and sequencing endogenously bound Mamu-B*01 ligands. We further characterized the peptide-binding characteristics using panels of single amino acid substitution analogs. Using this detailed motif, 507 peptides derived from SIV(mac)239 were identified and tested for their Mamu-B*01 binding capacity. Surprisingly, only 11 (2.2%) of these motif-containing peptides bound with IC50 values < or =500 nM. We assessed the immunogenicity of these peptides using freshly isolated PBMC from ten Mamu-B*01+ SIV-infected rhesus macaques in IFN-gamma ELISPOT and IFN-gamma/TNF-alpha intracellular cytokine staining assays. Lymphocytes from these SIV-infected macaques responded to none of these peptides. Furthermore, there was no sequence variation indicative of escape in the regions of the virus that encoded these peptides. Additionally, we could not confirm previous reports of SIV-derived Mamu-B*01-restricted epitopes in the Env and Gag proteins. Our results suggest that the high frequency MHC class I molecule, Mamu-B*01, is not involved in SIV-specific CD8+ T lymphocyte responses.

Alleles↗
PubMed2005

Rhesus macaque MHC class I molecules present HLA-B-like peptides.

SIV-infected Indian rhesus macaques (Macaca mulatta) are an important animal model for humans infected with HIV. Understanding macaque (M. mulatta class I (Mamu)) MHC class I-peptide binding facilitates the comparison of SIV- and HIV-specific cellular immune responses. In this study, we characterized the endogenous peptide-binding properties of three Mamu-A (A*02, A*08, A*11) and three Mamu-B (B*01, B*03, B*12) class I molecules. Motif comparisons revealed that five of the six macaque class I molecules (A*02, A*08, A*11, B*01, and B*03) have peptide-binding motifs similar to those of human class I molecules. Of the 65 macaque endogenous peptide ligands that we sequenced by tandem mass spectroscopy, 5 were previously eluted from HLA class I molecules. Nonamers predominated among the individual ligands, and both the motifs and the individual ligands indicated P2, P9, and various ancillary anchors. Interestingly, peptide binding of the Mamu-A and Mamu-B molecules exhibited cross-species peptide-presentation overlap primarily with HLA-B molecules. Indeed, all of the macaque class I molecules appeared HLA-B-like in peptide presentation. Remarkably, the overlap in macaque- and HLA-peptide presentation occurred despite divergent class I peptide-binding grooves. Macaque and human class I differing by up to 42 aa (13-23%) within the alpha-1 and alpha-2 domains, including substantial divergence within specificity pockets A-F, bound the same endogenous peptide. Therefore, endogenous peptide characterization indicates that macaque class I molecules may be the functional equivalents of HLA-B molecules.

Amino Acid Motifs↗
PubMed2005

Automated generation and evaluation of specific MHC binding predictive tools: ARB matrix applications.

Prediction of which peptides can bind major histocompatibility complex (MHC) molecules is commonly used to assist in the identification of T cell epitopes. However, because of the large numbers of different MHC molecules of interest, each associated with different predictive tools, tool generation and evaluation can be a very resource intensive task. A methodology commonly used to predict MHC binding affinity is the matrix or linear coefficients method. Herein, we described Average Relative Binding (ARB) matrix methods that directly predict IC(50) values allowing combination of searches involving different peptide sizes and alleles into a single global prediction. A computer program was developed to automate the generation and evaluation of ARB predictive tools. Using an in-house MHC binding database, we generated a total of 85 and 13 MHC class I and class II matrices, respectively. Results from the automated evaluation of tool efficiency are presented. We anticipate that this automation framework will be generally applicable to the generation and evaluation of large numbers of MHC predictive methods and tools, and will be of value to centralize and rationalize the process of evaluation of MHC predictions. MHC binding predictions based on ARB matrices were made available at http://epitope.liai.org:8080/matrix web server.

Animals↗
PubMed2005

Characterization of the peptide-binding specificity of Mamu-A*11 results in the identification of SIV-derived epitopes and interspecies cross-reactivity.

The SIV-infected Indian rhesus macaque is the most established model of HIV infection, providing insight into pathogenesis and a system for testing novel vaccines. However, only a limited amount of information is available regarding the peptide-binding motifs and epitopes bound by their class I and class II MHC molecules. In this study, we utilized a library of over 1,000 different peptides and a high throughput MHC-peptide binding assay to detail the binding specificity of the rhesus macaque class I molecule Mamu-A*11. These studies defined the fine specificity of primary anchor positions, and dissected the role of secondary anchors, for peptides of 8-11 residues in length. This detailed information was utilized to develop size-specific polynomial algorithms to predict Mamu-A*11 binding capacity. Testing SIVmac239-derived Mamu-A*11 binding peptides for recognition by peripheral blood mononuclear cells (PBMC) from Mamu-A*11-positive, SIV-infected macaques, identified five novel SIV-derived Mamu-A*11 epitopes. Finally, we detected extensive cross-reactivity at the binding level between Mamu-A*11 and the mouse H-2 class I molecule Kk. Further experiments revealed that three out of four Mamu-A*11 binding peptides which bound Kk and were immunogenic in Kk mice were also recognized in Mamu-A*11-infected macaques. This is the first detailed description of mouse-macaque interspecies cross-reactivity, potentially useful in testing novel vaccines in mice and macaques.

Amino Acid Sequence↗
PubMed2005

Unparalleled complexity of the MHC class I region in rhesus macaques.

The highly polymorphic gene products of the classical MHC class I genes in humans (HLA-A, HLA-B, and HLA-C) play a critical role in the immune defense against intracellular infections. Because non-human primates are important models for AIDS vaccine research, rhesus monkeys from a thoroughly pedigreed and serotyped colony were subjected to full-length cDNA analysis of MHC class I gene transcripts. Rhesus macaques express multiple dominant Mamu-A and Mamu-B transcripts (majors) per chromosome, which are characterized by high expression levels. The presence of additional cDNAs with low levels of expression (minors) suggests evidence for transcriptional control of MHC class I genes. Moreover, phylogenetic analyses illustrate that most of the Mamu-A and Mamu-B loci/lineages identified display no or only limited levels of allelic polymorphism. Thus, MHC class I diversity in rhesus macaques is typified by the existence of an unmatched high number of Mamu-A and Mamu-B region configurations that exhibit polymorphism with regard to the number and combination of transcribed loci present per chromosome.

Animals↗
PubMed2005

MHC polymorphism: AIDS susceptibility in non-human primates.

One of the main host factors controlling resistance to disease appears to be the MHC. The recent poor results in HIV-1/AIDS Phase 3 vaccine field trials underline the importance of non-human primate models for AIDS. These models have been, and will continue to be, important for the definition of protective immune responses relevant to successful vaccine design because they supply essential information on the basic biology of lentivirus infections, mechanisms of resistance, escape and vaccine development.

Acquired Immunodeficiency Syndrome↗
PubMed2005

The pigtail macaque MHC class I allele Mane-A*10 presents an immundominant SIV Gag epitope: identification, tetramer development and implications of immune escape and reversion.

The pigtail macaque (Macaca nemestrina) is a common model for the study of AIDS. The pigtail major histocompatibility complex class I allele Mane-A*10 restricts an immunodominant simian immunodeficiency virus (SIV) Gag epitope (KP9) which rapidly mutates to escape T cell recognition following acute simian/human immunodeficiency virus infection. Two technologies for the detection of Mane-A*10 in outbred pigtail macaques were developed: reference strand-mediated conformational analysis and sequence-specific primer polymerase chain reaction. A Mane-A*10/KP9 tetramer was then developed to quantify CD8(+) T lymphocytes primed by multigenic DNA vaccination, which have previously been difficult to detect using standard interferon-gamma-based T cell assays. We also demonstrate mutational escape at KP9 following acute SIV infection. Mane-A*10(+) animals have lower set point SIV levels than Mane-A*10(-) animals, suggesting a significant fitness cost of escape. These studies pave the way for a more robust understanding of HIV vaccines in pigtail macaques.

Alleles↗