Plasma RNA viral load as measured by the branched DNA and nucleic acid sequence-based amplification assays of HIV-1 subtypes A and D in Uganda.
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DNA, Viral↗
Biomedical subjects
B D Walker
Publications and source records attributed to B D Walker.
Persistent HIV-1-specific CTL clonal expansion despite high viral burden post in utero HIV-1 infection.
To address the issue of clonal exhaustion in humans, we monitored HLA class I-restricted, epitope-specific CTL responses in an in utero HIV-1-infected infant from 3 mo through 5 years of age. Serial functional CTL precursor assays demonstrated persistent, vigorous, and broadly directed HIV-1 specific CTL activity with a dominant response against an epitope in HIV-1 Gag-p17 (SLYNTVATL, aa 77-85). A clonal CTL response directed against the immunodominant, HLA-A*0201-restricted epitope was found to persist over the entire observation period, as shown by TCR analysis of cDNA libraries generated from PBMC. The analysis of autologous viral sequences did not reveal any escape mutations within the targeted epitope, and viral load measurement indicated ongoing viral replication. Furthermore, inhibition of viral replication assays indicated that the epitope was properly processed from autologous viral protein. These data demonstrate that persistent exposure to high levels of viral Ag does not necessarily lead to clonal exhaustion and that epitope-specific clonal CTL responses induced within the first weeks of life can persist for years without inducing detectable viral escape variants.
Child, Preschool↗
Acute HIV infection among patients tested for mononucleosis.
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Acute Disease↗
Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication.
It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.
Animals↗
Coalescent estimates of HIV-1 generation time in vivo.
The generation time of HIV Type 1 (HIV-1) in vivo has previously been estimated using a mathematical model of viral dynamics and was found to be on the order of one to two days per generation. Here, we describe a new method based on coalescence theory that allows the estimate of generation times to be derived by using nucleotide sequence data and a reconstructed genealogy of sequences obtained over time. The method is applied to sequences obtained from a long-term nonprogressing individual at five sampling occasions. The estimate of viral generation time using the coalescent method is 1.2 days per generation and is close to that obtained by mathematical modeling (1.8 days per generation), thus strengthening confidence in estimates of a short viral generation time. Apart from the estimation of relevant parameters relating to viral dynamics, coalescent modeling also allows us to simulate the evolutionary behavior of samples of sequences obtained over time.
DNA, Viral↗
Characterization of HIV-1-specific T-helper cells in acute and chronic infection.
HIV-1 infection is associated with progressive and relentless destruction of the immune system in the majority of infected persons, but some persons appear to be able to successfully contain the virus in the absence of antiviral therapy. Such cases suggest that the host immune response can successfully contain the virus, and provide the rationale for concerted efforts to understand the host immune response to the virus and to develop new strategies to combat the infection with immune based therapies. Historically, the greatest hole in the immune repertoire in HIV-1 infection has been the lack of strong virus-specific proliferative responses. However, new studies have identified a potent Th cell response in some infected persons, and have shown a statistically significant negative correlation between plasma viremia and virus-specific CD4 T-helper cells directed at the p24 protein. Moreover, early institution of potent antiviral therapy in the earliest stages of acute HIV-1 infection have led to persistent, strong HIV-1-specific T-helper cell responses, analogous to those seen in persons who are able to control viremia in the absence of antiviral therapy. We hypothesize that this is because potent antiviral therapy is able to protect virus-specific Th cells as they become activated, and thus these cells are not lost in the earliest stages of infection.
Acute Disease↗
T lymphocyte responses in HIV-1 infection: implications for vaccine development.
Substantial progress has been made over the past year in understanding the cellular immune response in HIV pathogenesis. Cytotoxic T lymphocytes play a critical role in establishing the level of viremia and virus-specific Th cell responses appear to affect the in vivo efficacy of cytotoxic T lymphocytes. Together, these new data provide important insights to refocus efforts aimed at immunotherapeutic interventions and vaccine development.
AIDS Vaccines↗
The great escape - AIDS viruses and immune control.
Many studies have been designed to address the role of CTL immune escape in HIV-1 infection, but have not given conclusive answers. Now, an elegant longitudinal analysis clearly demonstrates that progression to disease in SIV-infected macaques is associated with evasion of the CTL response (pages 1270-1276).
Animals↗
Inhibition of HERG channels stably expressed in a mammalian cell line by the antianginal agent perhexiline maleate.
Perhexiline has been used as an anti-anginal agent for over 25 years, and is known to cause QT prolongation and torsades de pointes. We hypothesized that the cellular basis for these effects was blockade of I(Kr). A stable transfection of HERG into a CHO-K1 cell line produced a delayed rectifier, potassium channel with similar properties to those reported for transient expression in Xenopus oocytes. Perhexiline caused voltage- and frequency-dependent block of HERG (IC50 7.8 microM). The rate of inactivation was increased and there was a 10 mV hyperpolarizing shift in the voltage-dependence of steady-state inactivation, suggestive of binding to the inactivated state. In conclusion, perhexiline potently inhibits transfected HERG channels and this is the probable mechanism for QT prolongation and torsades de pointes. Channel blockade shows greatest affinity for the inactivated state.
Algorithms↗
Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells.
1. The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. 2. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n-3 polyunsaturated fatty acids (n-3 PUFA) present in fish oils. 3. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. 4. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8 +/- 0.2 and 5.3 +/- 0.4 ms for activation and 20.0 +/- 1.2 and 96.6+/-6.7 ms for inactivation at +45 mV at 23 degrees C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation= -6.7+/-2.5 mV, V1,2 of steady state inactivation= -51.3+/-0.2 mV at 23 degrees C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37 degrees C) compared to room temperature (23 degrees C). 5. The n-3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 micromol L(-1). Blockade of the transient outward current may be an important mechanism by which n-3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.
Animals↗
Inhibition of the human ether-a-go-go-related gene (HERG) potassium channel by cisapride: affinity for open and inactivated states.
1 Cisapride is a prokinetic agent which has been associated with QT prolongation, torsades de pointes and cardiac arrest. The cellular mechanism for these observations is high affinity blockade of IKr (encoded by HERG). 2 In a chronic transfection model using CHO-K1 cells, cisapride inhibited HERG tail currents after a step to +25 mV with similar potency at room and physiological temperatures (IC50 16. 4 nM at 20-22 degrees C and 23.6 nM at 37 degrees C). 3 Channel inhibition exhibited time-, voltage- and frequency-dependence. In an envelope of tails test, channel blockade increased from 27+/-8% after a 120 ms depolarizing step to 50+/-4% after a 1.0 s step. These findings suggested affinity for open and/or inactivated channel states. 4 Inactivation was significantly accelerated by cisapride in a concentration-dependent manner and there was a small (-7 mV) shift in the voltage dependence of steady state inactivation. 5 Channel blockade by cisapride was modulated by [K+]o, with a 26% reduction in the potency of channel blockade when [K+]o was increased from 1 to 10 mM. 6 In conclusion, HERG channel inhibition by cisapride exhibits features consistent with open and inactivated state binding and is sensitive to external potassium concentration. These features may have significant clinical implications with regard to the mechanism and treatment of cisapride-induced proarrhythmia.
Animals↗
Improved CD4 lymphocyte outgrowth in response to effective antiretroviral therapy.
CD4 lymphocyte regenerative capacity was evaluated by use of an ex vivo outgrowth assay in human immunodeficiency virus (HIV)-1-infected subjects enrolled in a clinical trial (Merck 039). CD4 lymphocytes were selectively expanded in vitro by T cell receptor triggering, which also induces HIV production from latently infected cells. CD4 cell expansion and lack of virus production in cultures correlated well with clinical responses and were best in those receiving an aggressive antiretroviral three-drug regimen. Twelve clinical responders receiving triple-drug therapy monitored for 60 weeks had both excellent ex vivo CD4 cell expansion and lack of HIV replication, often in the absence of added drug in culture. Breakthrough viruses recovered from drug-containing arms of the cultures showed phenotypic resistance to the drugs used in vivo. This CD4 lymphocyte outgrowth assay correlates well with clinical outcome in subjects receiving potent antiretroviral regimens and may predict the emergence of early drug resistance.
Anti-HIV Agents↗
Immune reconstitution: is there a potential role for Filgrastim (r-metHuG-CSF).
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Acquired Immunodeficiency Syndrome↗
Recurrent atrial electrode displacement complicating a single lead VDD pacing system.
We describe an unusual complication of single-lead VDD pacing: recurrent advancement of the atrial bipole into the right ventricle. As a consequence, the patient experienced symptomatic pacemaker-mediated tachycardia and underwent two revision operations to achieve adequate fixation of the lead.
Electrocardiography↗
Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences.
Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.
Adolescent↗
Frequent detection of escape from cytotoxic T-lymphocyte recognition in perinatal human immunodeficiency virus (HIV) type 1 transmission: the ariel project for the prevention of transmission of HIV from mother to infant.
Host immunologic factors, including human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL), are thought to contribute to the control of HIV type 1 (HIV-1) replication and thus delay disease progression in infected individuals. Host immunologic factors are also likely to influence perinatal transmission of HIV-1 from infected mother to infant. In this study, the potential role of CTL in modulating HIV-1 transmission from mother to infant was examined in 11 HIV-1-infected mothers, 3 of whom transmitted virus to their offspring. Frequencies of HIV-1-specific human leukocyte antigen class I-restricted CTL responses and viral epitope amino acid sequence variation were determined in the mothers and their infected infants. Maternal HIV-1-specific CTL clones were derived from each of the HIV-1-infected pregnant women. Amino acid substitutions within the targeted CTL epitopes were more frequently identified in transmitting mothers than in nontransmitting mothers, and immune escape from CTL recognition was detected in all three transmitting mothers but in only one of eight nontransmitting mothers. The majority of viral sequences obtained from the HIV-1-infected infant blood samples were susceptible to maternal CTL. These findings demonstrate that epitope amino acid sequence variation and escape from CTL recognition occur more frequently in mothers that transmit HIV-1 to their infants than in those who do not. However, the transmitted virus can be a CTL susceptible form, suggesting inadequate in vivo immune control.
Base Sequence↗
Enhanced inhibition of human immunodeficiency virus type 1 by Met-stromal-derived factor 1beta correlates with down-modulation of CXCR4.
CXCR4 is a chemokine receptor used by some strains of HIV-1 as an entry coreceptor in association with cell surface CD4 on human cells. In human immunodeficiency virus type 1 (HIV-1)-infected individuals, the appearance of viral isolates with a tropism for CXCR4 (T tropic) has been correlated with late disease progression. The presumed natural ligands for CXCR4 are SDF-1alpha and SDF-1beta, which are proposed to play a role in blocking T-tropic HIV-1 cell entry. Here, we demonstrate that addition of an N-terminal methionine residue to SDF-1beta (Met-SDF-1beta) results in a dramatically enhanced functional activity compared to that of native SDF-1beta. Equivalent concentrations of Met-SDF-1beta are markedly more inhibitory for T-tropic HIV-1 replication than SDF-1beta. A comparison of the biological activities of these two forms of SDF-1beta reveals that Met-SDF-1beta induces a more pronounced intracellular calcium flux yet binds with slightly lower affinity to CXCR4 than SDF-1beta. Down-modulation of CXCR4 is similar after exposure of cells to either chemokine form for 2 h. However, after a 48-h incubation, the surface expression of CXCR4 is much lower for cells treated with Met-SDF-1beta. The enhanced blocking of T-tropic HIV-1 by Met-SDF-1beta appears to be related to prolonged CXCR4 down-modulation.
Amino Acid Sequence↗
Lack of viral escape and defective in vivo activation of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes in rapidly progressive infection.
Human immunodeficiency virus type 1 (HIV-1)-specific immune responses over the course of rapidly progressive infection are not well defined. Detailed longitudinal analyses of neutralizing antibodies, lymphocyte proliferation, in vivo-activated and memory cytotoxic T-lymphocyte (CTL) responses, and viral sequence variation were performed on a patient who presented with acute HIV-1 infection, developed an AIDS-defining illness 13 months later, and died 45 months after presentation. Neutralizing-antibody responses remained weak throughout, and no HIV-1-specific lymphocyte proliferative responses were seen even early in the disease course. Strong in vivo-activated CTL directed against Env and Pol epitopes were present at the time of the initial drop in viremia but were quickly lost. Memory CTL against Env and Pol epitopes were detected throughout the course of infection; however, these CTL were not activated in vivo. Despite an initially narrow CTL response, new epitopes were not targeted as the disease progressed. Viral sequencing showed the emergence of variants within the two targeted CTL epitopes; however, viral variants within the immunodominant Env epitope were well recognized by CTL, and there was no evidence of viral escape from immune system detection within this epitope. These data demonstrate a narrowly directed, static CTL response in a patient with rapidly progressive disease. We also show that disease progression can occur in the presence of persistent memory CTL recognition of autologous epitopes and in the absence of detectable escape from CTL responses, consistent with an in vivo defect in activation of CTL.
Adult↗