Biomedical subjects

Marcus Altfeld

Publications and source records attributed to Marcus Altfeld.

At least 55 records · Page 3Linked to original sources

PubMed2004

Immune selection for altered antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection.

Mutations within cytotoxic T lymphocyte (CTL) epitopes impair T cell recognition, but escape mutations arising in flanking regions that alter antigen processing have not been defined in natural human infections. In human histocompatibility leukocyte antigen (HLA)-B57+ HIV-infected persons, immune selection pressure leads to a mutation from alanine to proline at Gag residue 146 immediately preceding the NH2 terminus of a dominant HLA-B57-restricted epitope, ISPRTLNAW. Although N-extended wild-type or mutant peptides remained well-recognized, mutant virus-infected CD4 T cells failed to be recognized by the same CTL clones. The A146P mutation prevented NH2-terminal trimming of the optimal epitope by the endoplasmic reticulum aminopeptidase I. These results demonstrate that allele-associated sequence variation within the flanking region of CTL epitopes can alter antigen processing. Identifying such mutations is of major relevance in the construction of vaccine sequences.

Alleles↗

PubMed2004

HIV-1-specific cytotoxicity is preferentially mediated by a subset of CD8(+) T cells producing both interferon-gamma and tumor necrosis factor-alpha.

CD8(+) T cells play a crucial role in the control of viral infections by direct elimination of infected cells and secretion of a number of soluble factors. Recent data suggest that HIV-1-specific CD8(+) T cell subsets may differ in their ability to exert these effector functions. Here, we directly compared the cytokine secretion patterns and cytotoxic capacity of HIV-1-specific CD8(+) T cells, using a flow-cytometric cytotoxicity assay based on caspase-3 activation in dying target cells. These experiments revealed considerable intraindividual and interindividual differences among epitope-specific T-cell effector functions: while the frequency of HIV-1-specific CD8(+) T cells secreting interferon-gamma but no tumor necrosis factor-alpha (TNF-alpha) following antigenic stimulation was only weakly correlated to their cytotoxic activity (R = 0.05, P =.57), a subset of CD8(+) T cells secreting both inter-feron-gamma and TNF-alpha was substantially more strongly associated with cytotoxicity (R = 0.67, P <.001). This subset of CD8(+) T cells also exhibited stronger intracellular perforin expression and more pronounced direct ex vivo HIV-1-specific cytoxicity than CD8(+) T cells secreting solely interferon-gamma following sorting of these subpopulations according to their cytokine profile. These results suggest that HIV-1-specific cytotoxicity of CD8(+) T cells is preferentially mediated by a subset of CD8(+) T cells secreting both interferon-gamma and TNF-alpha.

Adult↗

PubMed2004

HIV-1 specific CD8+ T cells with an effector phenotype and control of viral replication.

Most people infected with HIV-1 cannot control viral replication despite the presence of virus-specific CD8+ T cells. It has been postulated that this inability is related to the failure of these cells to mature into fully differentiated effector cells. We tested this hypothesis by comparing the maturation phenotype of virus-specific CD8+ T cells in people who could control viral replication off anti-retroviral therapy with those who could not. In five patients with treated acute HIV-1-infection, structured treatment interruption (STI) induced control of viral replication was associated with expansion of virus-specific CD8+ T cells with a fully differentiated effector phenotype. These effector cells were also expanded in treatment-naive chronically infected individuals who spontaneously controlled viral replication, and augmented expression of perforin was noted in both settings. Our data show that full maturation of virus-specific CD8+ T cells is possible in the context of HIV-1-infection, and suggest that such maturation might be important in viral control.

Antiretroviral Therapy, Highly Active↗

PubMed2004

CD107a as a functional marker for the identification of natural killer cell activity.

Natural killer (NK) cells are a subset of lymphocytes that play a central role in the innate immune response to tumors and infections. An important limitation in the field of NK research is attributable to the deficit of assays available for the detection of the functional activity of NK cells. Recently, lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) has been described as a marker of CD8+ T-cell degranulation following stimulation. Here we describe CD107a as a marker of NK cell functional activity using multi-parameter flow cytometry. CD107a is significantly upregulated on the surface of NK cells following stimulation with MHC devoid targets. Additionally, CD107a expression correlates with both cytokine secretion and NK cell-mediated lysis of target cells. However, as well as being coordinately expressed on nearly all cytokine secreting cells, CD107a was also expressed on a large subset of NK cells that did not secrete cytokine following stimulation. These data suggest that employing CD107a as a marker of NK cell functional activity may allow for the identification of a large fraction of activated NK cells that may degranulate in the absence of cytokine secretion. Cumulatively, the data presented here demonstrate that CD107a is a sensitive marker of NK cell activity.

Antigens, CD↗

PubMed2004

Differences in the expressed HLA class I alleles effect the differential clustering of HIV type 1-specific T cell responses in infected Chinese and caucasians.

China is a region of the world with a rapidly spreading HIV-1 epidemic. Studies providing insights into HIV-1 pathogenesis in infected Chinese are urgently needed to support the design and testing of an effective HIV-1 vaccine for this population. HIV-1-specific T cell responses were characterized in 32 HIV-1-infected individuals of Chinese origin and compared to 34 infected caucasians using 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence in an IFN-gamma ELISpot assay. All HIV-1 proteins were targeted with similar frequency in both populations and all study subjects recognized at least one overlapping peptide. HIV-1-specific T cell responses clustered in seven different regions of the HIV-1 genome in the Chinese cohort and in nine different regions in the caucasian cohort. The dominant HLA class I alleles expressed in the two populations differed significantly, and differences in epitope clustering pattern were shown to be influenced by differences in class I alleles that restrict immunodominant epitopes. These studies demonstrate that the clustering of HIV-1-specific T cell responses is influenced by the genetic HLA class I background in the study populations. The design and testing of candidate vaccines to fight the rapidly growing HIV-1 epidemic must therefore take the HLA genetics of the population into account as specific regions of the virus can be expected to be differentially targeted in ethnically diverse populations.

Alleles↗

PubMed2004

Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection.

Numerous studies now support that human immunodeficiency virus type 1 (HIV-1) evolution is influenced by immune selection pressure, with population studies showing an association between specific HLA alleles and mutations within defined cytotoxic T-lymphocyte epitopes. Here we combine sequence data and functional studies of CD8 T-cell responses to demonstrate that allele-specific immune pressures also select for mutations flanking CD8 epitopes that impair antigen processing. In persons expressing HLA-A3, we demonstrate consistent selection for a mutation in a C-terminal flanking residue of the normally immunodominant Gag KK9 epitope that prevents its processing and presentation, resulting in a rapid decline in the CD8 T-cell response. This single amino acid substitution also lies within a second HLA-A3-restricted epitope, with the mutation directly impairing recognition by CD8 T cells. Transmission of the mutation to subjects expressing HLA-A3 was shown to prevent the induction of normally immunodominant acute-phase responses to both epitopes. However, subsequent in vivo reversion of the mutation was coincident with delayed induction of new CD8 T-cell responses to both epitopes. These data demonstrate that mutations within the flanking region of an HIV-1 epitope can impair recognition by an established CD8 T-cell response and that transmission of these mutations alters the acute-phase CD8(+) T-cell response. Moreover, reversion of these mutations in the absence of the original immune pressure reveals the potential plasticity of immunologically selected evolutionary changes.

Amino Acid Sequence↗

PubMed2004

Comprehensive analysis of human immunodeficiency virus type 1-specific CD4 responses reveals marked immunodominance of gag and nef and the presence of broadly recognized peptides.

Increasing evidence suggests that human immunodeficiency virus type 1 (HIV-1)-specific CD4 T-cell responses contribute to effective immune control of HIV-1 infection. However, the breadths and specificities of these responses have not been defined. We screened fresh CD8-depleted peripheral blood mononuclear cells (PBMC) from 36 subjects at different stages of HIV-1 infection for virus-specific CD4 responses by gamma interferon enzyme-linked immunospot assay, using 410 overlapping peptides spanning all HIV-1 proteins (based on the clade B consensus sequence). HIV-1-specific CD4 responses were identified in 30 of the 36 individuals studied, with the strongest and broadest responses detected in persons treated in acute infection who underwent treatment interruption. In individuals with identified responses, the total number of recognized HIV-1 peptides ranged from 1 to 36 (median, 7) and the total magnitude of responses ranged from 80 to >14,600 (median, 990) spot-forming cells/10(6) CD8-depleted PBMC. Neither the total magnitude nor the number of responses correlated with viremia. The most frequent and robust responses were directed against epitopes within the Gag and Nef proteins. Peptides targeted by >/=25% of individuals were then tested for binding to a panel of common HLA-DR molecules. All bound broadly to at least four of the eight alleles tested, and two bound to all of the HLA-DR molecules studied. Fine mapping and HLA restriction of the responses against four of these peptides showed a combination of clustering of epitopes and promiscuous presentation of the same epitopes by different HLA class II alleles. These findings have implications for the design of immunotherapeutic strategies and for testing candidate HIV vaccines.

Adult↗

PubMed2003

Influence of HLA-B57 on clinical presentation and viral control during acute HIV-1 infection.

BACKGROUND: HLA-B57, as well as cytotoxic T-lymphocyte (CTL) responses restricted by this allele, have been strongly associated with long-term non-progressive chronic HIV-1 infection. However, their impact on viral replication during acute HIV-1 infection is not known. METHODS: Clinical and immunological parameters during acute and early HIV-1 infection in individuals expressing HLA-B57 were assessed. HIV-1-specific T-cell responses were determined by peptide-specific interferon-gamma production measured using Elispot assay and flow-based intracellular cytokine quantification. RESULTS: Individuals expressing HLA-B57 presented significantly less frequently with symptomatic acute HIV-1 infection (4/116, 3.4%) than expected from the frequency of chronically infected individuals expressing this allele (43/446, 9.6%; P < 0.05). During acute infection, virus-specific CD8 T-cell responses were dominated by HLA-B57-restricted responses, with significantly broader (P < 0.02) and stronger (P < 0.03) responses restricted by HLA-B57 than restricted by all other co-expressed HLA class I alleles combined. Six out of nine individuals expressing HLA-B57 controlled HIV-1 viremia in the absence of therapy at levels < 5000 copies/ml (median, 515 copies/ml) during up to 29 months following acute infection. CONCLUSION: These data demonstrate that host genetic factors can influence the clinical manifestations of acute HIV-1 infection and provide a functional link between HLA-B57 and viral immune control.

Acute Disease↗

PubMed2003

Comparison of overlapping peptide sets for detection of antiviral CD8 and CD4 T cell responses.

Increasing efforts are directed towards the development of effective vaccines through induction of virus-specific T cell responses. Although emerging data indicate a significant role of these cells in determining viral set point in infections such as HIV, there is as yet no consensus as to the best methods for assaying the breadth of these responses. In this study, we used sensitive interferon gamma-based intracellular cytokine staining (ICS) and Elispot assays to determine the optimal overlapping peptide set to screen for these responses. Twenty persons with established HIV infection were studied, focusing on responses to the highly immunogenic Nef protein. Six different HIV-1 Nef peptide sets were used, ranging in length from 15 to 20 amino acids (aa), in overlap from 10 to 11 amino acids, and derived from two different B clade sequences. A total of 54 CD8 T cell responses to Nef peptides were found in this cohort, of which only 12 were detected using previously defined Nef optimal epitopes. No single peptide set detected all responses. Though there was a trend of the shorter peptides detecting more CD8 T cell responses than the 20 amino acid long peptides and longer peptides detecting more CD4 T cell responses, neither was statistically significant. There was no difference between an overlap of 10 or 11 amino acids. All responses detected with the six different sets of overlapping peptides were towards the more highly conserved regions of Nef. We conclude that peptides ranging from 15 to 20 amino acids yield similar results in IFN-gamma-based Elispot and ICS assays, and that all are likely to underestimate the true breadth of responses to a given reference strain of virus.

Amino Acid Sequence↗

PubMed2003

Viremia control despite escape from a rapid and potent autologous neutralizing antibody response after therapy cessation in an HIV-1-infected individual.

The neutralizing Ab response after primary HIV-1 infection is delayed relative to the virus-specific CD8(+) T cell response and the initial decline in plasma viremia. Because nearly all HIV-1 infections result in AIDS, it would be instructive to study cases where neutralizing Ab production commenced sooner. This was done in subject AC10, an individual treated during early infection and in whom a rapid autologous neutralizing Ab response was detected after therapy cessation as rebound viremia declined and remained below 1000 RNA copies/ml of blood for over 2.5 years. This subject's Abs were capable of reducing the infectivity of his rebound virus by >4 logs in vitro at a time when rebound viremia was down-regulated and virus-specific CD8(+) T cells were minimal, suggesting that neutralizing Abs played an important role in the early control of viremia. The rebound virus did not exhibit an unusual phenotype that might explain its high sensitivity to neutralization by autologous sera. Neutralization escape occurred within 75 days and was proceeded by neutralizing Ab production to the escape variant and subsequent escape. Notably, escape was not associated with a significant rise in plasma viremia, perhaps due to increasing CD8(+) T cell responses. Sequence analysis of gp160 revealed a growing number of mutations over time, suggesting ongoing viral evolution in the face of potent antiviral immune responses. We postulate that an early effective neutralizing Ab response can provide long-term clinical benefits despite neutralization escape.

Adult↗

PubMed2003

HIV-1 superinfection.

During the past year, a number of reports have described HIV-1 superinfection in human subjects, defined as the reinfection of an individual with a second heterologous strain of HIV-1. These reports have challenged the assumption that HIV-1-specific immune responses generated during primary infection are protective against subsequent infection and have raised concern, not only with respect to HIV-1-positive individuals engaging in unsafe sex but also from the standpoint of developing effective vaccines. Herein we review the published reports of HIV-1 superinfection and highlight studies providing additional insight into the potential for HIV-1 superinfections to affect the global epidemic.

Animals↗

PubMed2003

Rev activity determines sensitivity of HIV-1-infected primary T cells to CTL killing.

The HIV Nef protein is thought to promote HIV immune evasion by downmodulating MHC-I and protecting infected cells from CTL killing. In addition, we demonstrated that Rev, an HIV regulatory protein needed for expression of the HIV late genes, can influence CTL killing. When Rev activity level was reduced by virtue of amino acid alterations in the Rev protein sequence, infected cells were more resistant to anti-Gag and anti-Env CTL killing. A screen of primary viral isolates revealed that viruses derived from asymptomatic, infected people had lower Rev activity, lower Gag levels, and greater resistance to anti-Gag CTL killing. Thus, rev alleles with low activity may have a selective advantage in infected people with effective immune responses.

Alleles↗

PubMed2003

Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences.

The antigenic diversity of human immunodeficiency virus type 1 (HIV-1) represents a significant challenge for vaccine design as well as the comprehensive assessment of HIV-1-specific immune responses in infected persons. In this study we assessed the impact of antigen variability on the characterization of HIV-1-specific T-cell responses by using an HIV-1 database to determine the sequence variability at each position in all expressed HIV-1 proteins and a comprehensive data set of CD8 T-cell responses to a reference strain of HIV-1 in infected persons. Gamma interferon Elispot analysis of HIV-1 clade B-specific T-cell responses to 504 overlapping peptides spanning the entire expressed HIV-1 genome derived from 57 infected subjects demonstrated that the average amino acid variability within a peptide (entropy) was inversely correlated to the measured frequency at which the peptide was recognized (P = 6 x 10(-7)). Subsequent studies in six persons to assess T-cell responses against p24 Gag, Tat, and Vpr peptides based on autologous virus sequences demonstrated that 29% (12 of 42) of targeted peptides were only detected with peptides representing the autologous virus strain compared to the HIV-1 clade B consensus sequence. The use of autologous peptides also allowed the detection of significantly stronger HIV-1-specific T-cell responses in the more variable regulatory and accessory HIV-1 proteins Tat and Vpr (P = 0.007). Taken together, these data indicate that accurate assessment of T-cell responses directed against the more variable regulatory and accessory HIV-1 proteins requires reagents based on autologous virus sequences. They also demonstrate that CD8 T-cell responses to the variable HIV-1 proteins are more common than previously reported.

Amino Acid Sequence↗

PubMed2002

HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus.

Early treatment of acute HIV-1 infection followed by treatment interruptions has shown promise for enhancing immune control of infection. A subsequent loss of control, however, allows the correlates of protective immunity to be assessed. Here we show that sudden breakthrough of plasma viraemia occurred after prolonged immune containment in an individual infected with HIV-1 at a time when 25 distinct CD8+ T-cell epitopes in the viral proteins Gag, RT, Integrase, Env, Nef, Vpr, Vif and Rev were being targeted. Sequencing of the virus in plasma and cells showed that superinfection with a second clade-B virus was coincident with the loss of immune control. This sudden increase in viraemia was associated with a decline in half of the CD8+ T-cell responses. The declining CD8+ T-cell responses were coupled with sequence changes relative to the initial virus that resulted in impaired recognition. Our data show that HIV-1 superinfection can occur in the setting of a strong and broadly directed virus-specific CD8+ T-cell response. The lack of cross-protective immunity for closely related HIV-1 strains, despite persistent recognition of multiple CD8 epitopes, has important implications for public health and vaccine development.

Amino Acid Sequence↗

PubMed2002

HIV-1 Vpu represents a minor target for cytotoxic T lymphocytes in HIV-1-infection.

We have previously shown that Vpu is rarely targeted by HIV-1-specific cytotoxic T lymphocytes (CTL). The present report extends these findings and describes the characterization of the first CTL epitope within HIV-1 Vpu, identified in an individual with long-term non-progressive HIV-1 infection. The epitope was shown to be highly conserved among HIV clade B sequences and is restricted by HLA-A*3303, an HLA allele commonly seen in Asian and west-African populations.

Amino Acid Sequence↗

PubMed2002

Important contribution of p15 Gag-specific responses to the total Gag-specific CTL responses.

OBJECTIVES: HIV-1 p15 Gag and its protease cleavage products, NCp7 and p6, are believed to play a major role in viral infectivity and assembly during the early and late stages of the retroviral life cycle. However, the extent to which p15 Gag is targeted by the host immune system in natural infection as well as precise cytotoxic T lymphocyte (CTL) epitopes within this protein remains to be defined. METHODS: In this study, 57 HIV-1 infected individuals and 10 HIV-1 negative controls were screened for CD8 and CD4 T-cell responses using overlapping peptides spanning the entire p15 Gag protein as well as the p17 Gag and p24 Gag proteins. Peptide-specific interferon-gamma production was measured by Elispot assay and flow-based intracellular cytokine quantification, and cytotoxic activity was confirmed after isolation of peptide-specific CD8 T-cell lines. RESULTS: CD8 T lymphocytes specific to p15 Gag were found in 46% (26/57) of HIV-1 infected individuals studied and contributed on average 17% (range, 0-100%) to the total Gag-specific T-cell responses. Responses were clustered within three immunodominant regions of p15 Gag, mapping to important functional sites. These studies also include the description of the first three optimally defined CTL epitopes within p15 Gag. CONCLUSIONS: These results indicate that p15 Gag is frequently recognized by HIV-1-specific CD8 T cells in HIV-1 infection and will be important in the comprehensive assessments of CTL responses in infected persons, as well as the design and testing of future HIV-1 vaccines and immunotherapeutic interventions.

Amino Acid Sequence↗

PubMed2002

Consistent patterns in the development and immunodominance of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses following acute HIV-1 infection.

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses generated during acute infection play a critical role in the initial control of viremia. However, little is known about the viral T-cell epitopes targeted during acute infection or about their hierarchy in appearance and relative immunodominance over time. In this study, HIV-1-specific CD8+ T-cell responses in 18 acutely infected individuals expressing HLA-A3 and/or -B7 were characterized. Detailed analysis of CD8 responses in one such person who underwent treatment of acute infection followed by reexposure to HIV-1 through supervised treatment interruptions (STI) revealed recognition of only two cytotoxic T-lymphocyte (CTL) epitopes during symptomatic acute infection. HIV-1-specific CD8+ T-cell responses broadened significantly during subsequent exposure to the virus, ultimately targeting 27 distinct CTL epitopes, including 15 different CTL epitopes restricted by a single HLA class I allele (HLA-A3). The same few peptides were consistently targeted in an additional 17 persons expressing HLA-A3 and/or -B7 during acute infection. These studies demonstrate a consistent pattern in the development of epitope-specific responses restricted by a single HLA allele during acute HIV-1 infection, as well as persistence of the initial pattern of immunodominance during subsequent STI. In addition, they demonstrate that HIV-1-specific CD8+ T-cell responses can ultimately target a previously unexpected and unprecedented number of epitopes in a single infected individual, even though these are not detectable during the initial exposure to virus. These studies have important implications for vaccine design and evaluation.

Acute Disease↗

PubMed2002

Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation.

The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequences reveal that defined epitopes tend to cluster. Here we integrate the global sequence and immunology databases to systematically explore the relationship between HIV-1 amino acid sequences and CTL epitope distributions. CTL responses to five HIV-1 proteins, Gag p17, Gag p24, reverse transcriptase (RT), Env, and Nef, have been particularly well characterized in the literature to date. Through comparing CTL epitope distributions in these five proteins to global protein sequence alignments, we identified distinct characteristics of HIV amino acid sequences that correlate with CTL epitope localization. First, experimentally defined HIV CTL epitopes are concentrated in relatively conserved regions. Second, the highly variable regions that lack epitopes bear cumulative evidence of past immune escape that may make them relatively refractive to CTLs: a paucity of predicted proteasome processing sites and an enrichment for amino acids that do not serve as C-terminal anchor residues. Finally, CTL epitopes are more highly concentrated in alpha-helical regions of proteins. Based on amino acid sequence characteristics, in a blinded fashion, we predicted regions in HIV regulatory and accessory proteins that would be likely to contain CTL epitopes; these predictions were then validated by comparison to new sets of experimentally defined epitopes in HIV-1 Rev, Tat, Vif, and Vpr.

Amino Acid Sequence↗