Currently, few rodent models of AIDS-associated non-Hodgkin’s lymphoma (AIDS-NHL) exist. In these studies, a novel mouse/human xenograft model of AIDS-associated Burkitt lymphoma (AIDS-BL) was created by injecting cells of the human AIDS-BL cell line, 2F7, intraperitoneally into NOD-SCID mice. Mice developed tumors in the peritoneal cavity, with metastases to the spleen, thymus, and mesenteric lymph nodes. Expression of the chemokine receptor, CXCR5, was greatly elevated in vivo on BL tumor cells in this model, as shown by flow cytometry. CXCL13 is the ligand for CXCR5, and serum and ascites levels of murine, but not human, CXCL13 showed a striking elevation in tumor-bearing mice, with levels as high as 200,000 pg/ml in ascites, as measured by ELISA. As shown by immunohistochemistry, murine CXCL13 was associated with macrophage-like tumor-infiltrating cells that appeared to be histiocytes. Blocking CXCR5 on 2F7 cells with neutralizing antibodies prior to injection into the mice substantially delayed tumor formation. The marked elevations in tumor cell CXCR5 expression and in murine CXCL13 levels seen in the model may potentially identify an important link between tumor-interacting histiocytes and tumor cells in AIDS-BL. These results also identify CXCL13 as a potential biomarker for this disease, which is consistent with previous studies showing that serum levels of CXCL13 were elevated in human subjects who developed AIDS-lymphoma. This mouse model may be useful for future studies on the interactions of the innate immune system and AIDS-BL tumor cells, as well as for the assessment of potential tumor biomarkers for this disease.
Recent research in autism spectrum disorder (ASD) has aroused interest in anterior cingulate cortex and in the neurometabolite glutamate. We report two studies of pregenual anterior cingulate cortex (pACC) in pediatric ASD. First, we acquired in vivo single-voxel proton magnetic resonance spectroscopy (1H MRS) in 8 children with ASD and 10 typically developing controls who were well matched for age, but with fewer males and higher IQ. In the ASD group in midline pACC, we found mean 17.7% elevation of glutamate + glutamine (Glx) (p<0.05) and 21.2% (p<0.001) decrement in creatine + phosphocreatine (Cr). We then performed a larger (26 subjects with ASD, 16 controls) follow-up study in samples now matched for age, gender, and IQ using proton magnetic resonance spectroscopic imaging (1H MRSI). Higher spatial resolution enabled bilateral pACC acquisition. Significant effects were restricted to right pACC where Glx (9.5%, p<0.05), Cr (6.7%, p<0.05), and N-acetyl-aspartate + N-acetyl-aspartyl-glutamate (10.2%, p<0.01) in the ASD sample were elevated above control. These two independent studies suggest hyperglutamatergia and other neurometabolic abnormalities in pACC in ASD, with possible right-lateralization. The hyperglutamatergic state may reflect an imbalance of excitation over inhibition in the brain as proposed in recent neurodevelopmental models of ASD.
CD4+ T cells and macrophages are the primary target cells for HIV in vivo, and antiretroviral drugs can vary in their ability to inhibit the infection of these different cell types. Resistance pathways to the HIV integrase inhibitor raltegravir have previously been investigated in T cells. Primary raltegravir resistance mutations, most often at integrase amino acid position 148 or 155, afford some resistance to the drug. The acquisition of pathway-specific secondary mutations then provides higher-level resistance to viruses infecting T cells. We show here that during macrophage infection, the presence of a single primary raltegravir resistance mutation (Q148H, Q148R, N155H, or N155S) is sufficient to provide resistance to raltegravir comparable to that seen in viruses expressing both primary and secondary mutations in costimulated CD4+ T cells. These data implicate macrophages as a potential in vivo reservoir that may facilitate the development of resistance to raltegravir. Notably, the newer integrase inhibitor MK-2048 effectively suppressed the infection of all raltegravir-resistant viruses in both T cells and macrophages, indicating that more recently developed integrase inhibitors are capable of inhibiting infection in both major HIV cellular reservoirs, even in patients harboring raltegravir-resistant viruses.
Osteoporosis, which contributes to morbidity and mortality, often coexists with cardiovascular disease, especially atherosclerosis. We have reported recently that in vitro exposure of human T-lymphocytes to oxidized lipids induced expression of a key osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL). Our previous studies have shown that mice fed an atherogenic high-fat diet developed osteopenia and that bone marrow preosteoclasts from these hyperlipidemic mice have increased osteoclastic potential. To investigate the role of T-lymphocytes in the diet-induced bone loss, C57BL/6 mice were fed either chow or a high-fat diet, and bone parameters and T-lymphocyte activation were assessed at 6 and 11 months. Consistent with our previous findings, peripheral quantitative computed tomographic (pQCT) analysis showed that mice in the high-fat group had lower bone mineral content than mice in the chow group. Furthermore, histomorphometric analysis showed decreased structural parameters in the high-fat group. Coculture studies showed that bone marrow cells isolated from the high-fat group, which contained increased levels of activated memory T-lymphocytes compared with bone marrow cells from the chow mice, supported osteoclastic differentiation of RAW 264.7 cells. Additionally, RANKL expression was upregulated significantly in the T-lymphocytes isolated from the bone marrow of the high-fat group. Splenic T-lymphocytes isolated from the high-fat group also had increased expression of transcripts for the receptor for oxidized lipids (LOX-1) as well as for inflammatory and osteoclastogenic cytokines, including RANKL, interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), IL-1β, and interferon γ (IFN-γ). Together these findings suggest that T-lymphocytes play a key role in the osteoclastogenesis induced by a high-fat diet and may contribute to the bone loss associated with diet-induced osteopenia. © 2010 American Society for Bone and Mineral Research.
T-lymphocytes; lipids; osteoporosis; cytokines; inflammation
The purpose of the study was to determine associations between pre-antiretroviral therapy (ART) senescent CD8+ T lymphocytes and naïve versus non-naive CD8+ and CD4+ T lymphocyte subpopulations and CD4+ responses after initiation of ART in younger versus older individuals.
Retrospective analysis of 100 subjects with pre-ART cryopreserved peripheral blood mononuclear cells samples was performed with flow cytometry. Subjects were divided into four groups by age (30–50 years or >50 years) and 96-week CD4+ response (<100 or >200 cells/mm3). All subjects had 96-week viral suppression to <50 copies/ml. Regression was utilized to investigate associations between pre-ART CD8+ and CD4+ T cell phenotypes with age and CD4+ response categories.
Individuals <50 years had a lower frequency of senescent CD8+ T lymphocytes of the CD56+57+, CD56+, and CD28− phenotypes (95%CI −3.6 to −0.02; 95%CI −4.2 to −0.03; 95%CI −12.5 to −1.4, respectively) and a higher frequency of naïve (CD45RA+CD28+) CD8+ T lymphocytes (95%CI 2.6 to 10.9). Younger age and good CD4+ response were associated with a higher frequency of pre-ART naïve CD4+ T cells (95%CI 2.0 to 16.4 and 95% CI 1.5 to 15.6, respectively).
Prior to ART, younger HIV-infected individuals have a higher frequency of naïve CD4+ and CD8+ T cells and lower frequency of senescent CD8+ T cell phenotypes.
HIV/AIDS; aging; immune risk phenotype; antiretroviral therapy; immune senescence
Characterization of residual plasma virus during antiretroviral therapy (ART) is a high priority to improve understanding of HIV-1 pathogenesis and therapy. To understand the evolution of HIV-1 pol and env genes in viremic patients under selective pressure of ART, we performed longitudinal analyses of plasma-derived pol and env sequences from single HIV-1 genomes. We tested the hypotheses that drug resistance in pol was unrelated to changes in coreceptor usage (tropism), and that recombination played a role in evolution of viral strains. Recombinants were identified by using Bayesian and other computational methods. High-level genotypic resistance was seen in ~70% of X4 and R5 strains during ART. There was no significant association between resistance and tropism. Each patient displayed at least one recombinant encompassing env and representing a change in predicted tropism. These data suggest that, in addition to mutation, recombination can play a significant role in shaping HIV-1 evolution.
HIV-1 drug resistance; HIV-1 recombination; HIV-1 tropism
The rates of immunologic and clinical progression are lower in patients with drug-resistant HIV compared to wild-type HIV. This difference is not fully explained by viral load. It has been argued that reductions in T cell activation and/or viral fitness might result in preserved target cells and an altered relationship between the level of viremia and the rate of CD4+ T cell loss. We tested this hypothesis over time in a cohort of patients with highly resistant HIV. Fifty-four antiretroviral-treated patients with multi-drug resistant HIV and detectable plasma HIV RNA were followed longitudinally. CD4+ T cell counts and HIV RNA levels were measured every 4 weeks and T cell activation (CD38/HLA-DR) was measured every 16 weeks. We found that the levels of CD4+ T cell activation over time were a strong independent predictor of CD4+ T cell counts while CD8+ T cell activation was more strongly associated with viremia. Using spectral analysis, we found strong evidence for oscillatory (or cyclic) behavior in CD4+ T cell counts, HIV RNA levels, and T cell activation. Each of the cell populations exhibited an oscillatory behavior with similar frequencies. Collectively, these data suggest that there may be a mechanistic link between T cell activation, CD4+ T cell counts, and viremia and lends support for the hypothesis of altered predator-prey dynamics as a possible explanation of the stability of CD4+ T cell counts in the presence of sustained multi-drug resistant viremia.
In this study, we investigated the possibility of differential effects of protease inhibitor (PI)-containing (PI+) and PI-sparing (PI−) antiretroviral therapies (ART) on CD8+ T cell apoptosis. We retrospectively analyzed both PD-1 expression and CD8+ T cell apoptosis in a cross-sectional study of HIV-positive adolescents and young adults (mean age = 17.4 years), with perinatally or behaviorally acquired HIV infection. Fifty-one specimens of cryopreserved peripheral blood mononuclear cells (PBMCs) were analyzed using 7-color flow cytometry: 20 from patients receiving PI+ ART, 14 from PI− ART, and 17 from the untreated. The results showed that percentages of PD-1+ CD8+ T cells were strongly correlated with plasma viral loads regardless of treatment (p = 0.0001). The percentage of PD-1+ CD8+ T cells was also positively associated with percentages of Annexin V+ CD8+ T cells (p = 0.04) in the PI+-treated group. The fraction of apoptotic (Annexin V+) CD8+ T cells was associated with viral load in the patients receiving ART that contained one or more protease inhibitors (p = 0.029), but not in the PI− or untreated groups. In summary, we found a direct correlation between PD-1 expression on CD8+ T cells and HIV levels that was not affected by types of medications used in the ART of those adolescents, suggesting that virological success is necessary for PD-1 downregulation. CD8+ T cell apoptosis was linked to high levels of PD-1 expression and HIV viremia. However, there was a higher degree of apoptosis among viremic patients receiving PI therapy, suggesting an immunologically adverse effect of continuing PI+ therapy after virological failure.
Although some individuals who abuse methamphetamine have considerable cognitive deficits, no prior studies have examined whether neurocognitive functioning is associated with outcome of treatment for methamphetamine dependence. In an outpatient clinical trial of bupropion combined with cognitive behavioral therapy and contingency management (Shoptaw et al., 2008), 60 methamphetamine-dependent adults completed three tests of reaction time and working memory at baseline. Other variables that were collected at baseline included measures of drug use, mood/psychiatric functioning, employment, social context, legal status, and medical status. We evaluated the relative predictive value of all baseline measures for treatment outcome using Classification and Regression Trees (CART; Breiman, 1984), a nonparametric statistical technique that produces easily interpretable decision rules for classifying subjects that are particularly useful in clinical settings. Outcome measures were whether or not a participant completed the trial and whether or not most urine tests showed abstinence from methamphetamine abuse. Urine-verified methamphetamine abuse at the beginning of the study was the strongest predictor of treatment outcome; two psychosocial measures (e.g., nicotine dependence and Global Assessment of Functioning) also offered some predictive value. A few reaction time and working memory variables were related to treatment outcome, but these cognitive measures did not significantly aid prediction after adjusting for methamphetamine usage at the beginning of the study. On the basis of these findings, we recommend that research groups seeking to identify new predictors of treatment outcome compare the predictors to methamphetamine usage variables to assure that unique predictive power is attained.
methamphetamine; treatment; relapse; neuropsychology; cognitive; abstinence; retention; predictors; prediction; outcome
Osteoporosis is a systemic disease that is associated with increased morbidity, mortality and health care costs. Whereas osteoclasts and osteoblasts are the main regulators of bone homeostasis, recent studies underscore a key role for the immune system, particularly via activation-induced T lymphocyte production of Receptor Activator of NFκB Ligand (RANKL). Well-documented as a mediator of T lymphocyte/dendritic cell interactions, RANKL also stimulates the maturation and activation of bone-resorbing osteoclasts. Given that lipid oxidation products mediate inflammatory and metabolic disorders such as osteoporosis and atherosclerosis, and since oxidized lipids affect several T lymphocyte functions, we hypothesized that RANKL production might also be subject to modulation by oxidized lipids. Here, we show that short term exposure of both unstimulated and activated human T lymphocytes to minimally oxidized low density lipoprotein (LDL), but not native LDL, significantly enhances RANKL production and promotes expression of the lectin-like oxidized LDL receptor-1 (LOX-1). The effect, which is also observed with 8-iso-Prostaglandin E2, an inflammatory isoprostane produced by lipid peroxidation, is mediated via the NFκB pathway, and involves increased RANKL mRNA expression. The link between oxidized lipids and T lymphocytes is further reinforced by analysis of hyperlipidemic mice, in which bone loss is associated with increased RANKL mRNA in T lymphocytes and elevated RANKL serum levels. Our results suggest a novel pathway by which T lymphocytes contribute to bone changes, namely, via oxidized lipid enhancement of RANKL production. These findings may help elucidate clinical associations between cardiovascular disease and decreased bone mass, and may also lead to new immune-based approaches to osteoporosis.
T lymphocytes; RANKL; lipids; bone; osteoporosis; human; mouse
Background. The homeostatic chemokine, CXCL13 (BLC, BCA-1), helps direct the recirculation of mature, resting B cells, which express its receptor, CXCR5. CXCL13/CXCR5 are expressed, and may play a role, in some non-AIDS-associated B cell tumors. Objective. To determine if CXCL13/CXCR5 are associated with AIDS-related non-Hodgkin's lymphoma (AIDS-NHL). Methods. Serum CXCL13 levels were measured by ELISA in 46 subjects who developed AIDS-NHL in the Multicenter AIDS Cohort Study and in controls. The expression or function of CXCL13 and CXCR5 was examined on primary AIDS-NHL specimens or AIDS-NHL cell lines. Results. Serum CXCL13 levels were significantly elevated in the AIDS-NHL group compared to controls. All primary AIDS-NHL specimens showed CXCR5 expression and most also showed CXCL13 expression. AIDS-NHL cell lines expressed CXCR5 and showed chemotaxis towards CXCL13. Conclusions. CXCL13/CXCR5 are expressed in AIDS-NHL and could potentially be involved in its biology. CXCL13 may have potential as a biomarker for AIDS-NHL.
Enfuvirtide (ENF) is a fusion inhibitor that prevents the entry of HIV virions into target cells. Studying the characteristics of viral evolution during treatment and after a treatment interruption can lend insight into the mechanisms of viral evolution and fitness. Although interruption of anti-HIV therapy often results in rapid emergence of an archived “wild-type” virus population, previous work from our group indicates that when only ENF is interrupted, viral gp41 continues to evolve forward and resistance mutations are lost due to back-mutation and remodeling of the envelope protein. To examine the co-evolution of gp120 and gp41 during ENF interruption we extend the Bayesian Hierarchical Phylogenetic model (HPM). Current HPMs enforce conditional independence across all outcomes while biologically all gene regions within a patient should return the same tree unless recombination confers an evolutionary selective advantage. A two-way-interaction HPM is proposed that provides middle ground between these two extremes and allows us to test for differences in evolutionary pressures across gene regions in multiple patients simultaneously. When the model is applied to a well-characterized cohort of HIV-infected patients interrupting ENF we find that across patients, the virus continued to evolve forward in both gene regions. Overall, the hypothesis of independence over dependence between the gene regions is supported. Models that allow for the examination of co-evolution over time will be increasingly important as more therapeutic classes are developed, each of which may impact other through novel and complex mechanisms.
NK cells mediate the innate immune response, and HIV-infected individuals demonstrate altered NK cell phenotype and function. We find that CD4+ NK cells are susceptible to HIV infection; this could account for the NK cell dysfunction seen in HIV-infected individuals. CD4+ NK cells express CXCR4 and can be infected with X4-tropic viruses and some primary R5-utilizing viral isolates. Treatment with the CXCR4 ligands AMD3100 and SDF-1α partially blocks infection with X4-tropic virus, treatment with anti-CCL Igs upregulates CCR5 surface expression and enables infection with HIV-Bal. HIV infection of NK cells results in CD4 downregulation and the production of infectious virus. HIV-infected CD4+ NK cells mediate NK cell cytotoxicity, however, HIV infection is associated with decreased chemotaxis towards IL-16. Thus, HIV infection of CD4+ NK cells could account for the NK cell dysfunction observed in HIV-infected individuals. Furthermore infected NK cells could serve as a viral reservoir of HIV in vivo.
NK cells; HIV; CD4 expression; CD4 downregulation; chemotaxis
Although antiretroviral drug resistance is common in treated HIV infected individuals, it is not a consistent indicator of HIV morbidity and mortality. To the contrary, HIV resistance-associated mutations may lead to changes in viral fitness that are beneficial to infected individuals. Using a bioinformatics-based model to assess the effects of numerous drug resistance mutations, we determined that the D30N mutation in HIV-1 protease had the largest decrease in replication capacity among known protease resistance mutations. To test this in silico result in an in vivo environment, we constructed several drug-resistant mutant HIV-1 strains and compared their relative fitness utilizing the SCID-hu mouse model. We found HIV-1 containing the D30N mutation had a significant defect in vivo, showing impaired replication kinetics and a decreased ability to deplete CD4+ thymocytes, compared to the wild-type or virus without the D30N mutation. In comparison, virus containing the M184V mutation in reverse transcriptase, which shows decreased replication capacity in vitro, did not have an effect on viral fitness in vivo. Thus, in this study we have verified an in silico bioinformatics result with a biological assessment to identify a unique mutation in HIV-1 that has a significant fitness defect in vivo.
HIV-1; replication capacity; bioinformatics; Bayesian; variable selection; exchangeable on subsets; prior model selection; in vivo validation.
Our aim was to elucidate the mechanism by which HIV transmission is increased following obstetrical hemorrhage. We investigated whether fetal allostimulation of maternal cells, which could occur following fetal-to-maternal hemorrhage, increases proliferation, HIV replication, and cellular activation. Peripheral blood mononuclear cells (PBMCs) were collected from HIV-infected mothers and their infants to assess maternal-fetal allostimulation. Responses were compared to allostimulation with unrelated donors. Maternal and fetal cells were cocultured to assess allogeneic stimulation. Cell proliferation was measured by [3H]thymidine incorporation and cell activation was assessed via fluorochrome-labeled antibody staining and flow cytometric analysis. Virus production from HIV-infected maternal cells was quantitated by p24 enzyme-linked immunosorbent assay or by branched chain DNA assay. Allostimulation with fetal cells led to maternal cell proliferation. In women with unsuppressed viral loads, virus release was also enhanced following allostimulation of maternal cells with fetal cells. Fetal cells are capable of allogeneically stimulating maternal cells, with responses comparable to those seen following allostimulation with unrelated donors. Allostimulation of maternal cells by fetal cells results in statistically significant increases in proliferation and enhanced HIV replication, suggesting a possible physiological mechanism for mother-to-child transmission of HIV in women with obstetrical hemorrhage.
Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8+ CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8+ T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection.
Marijuana smoking is associated with a number of abnormal findings in the lungs of habitual smokers. Previous studies revealed that Δ9-tetrahydrocannabinol (THC) caused mitochondrial injury in primary lung epithelial cells and in the cell line, A549 (Sarafian et al., 2003; Sarafian et al., 2005). The role of cannabinoid receptors in this injury was unclear, as was the potential impact on cell function. In order to investigate these questions, A549 cells were engineered to over-express the type 2 cannabinoid receptor (CB2R) using a self-inactivating lentiviral vector. This transduction resulted in a 60-fold increase in CB2R mRNA relative to cells transduced with a control vector. Transduced cell lines were used to study the effects of THC on chemotactic activity and mitochondrial function. Chemotaxis in response to a 10 % serum gradient was suppressed in a concentration-dependent manner by exposure to THC. CB2R-transduced cells exhibited less intrinsic chemotactic activity (p < 0.05) and were 80- to 100-fold more sensitive to the inhibitory effects of THC. Studies using SR144528, a selective CB2R antagonist, verified that these effects were mediated by the CB2R. Marijuana smoke extract, but not smoke extracts from tobacco or placebo marijuana cigarettes, reproduced these effects (p < 0.05). THC decreased ATP level and mitochondrial membrane potential (Ψm) in both control and CB2R-transduced cells. However, these decreases did not play a significant role in chemotaxis inhibition since cyclosporine A, which protected against ATP loss, did not increase cell migration. Moreover, CB2R-transduced cells displayed higher Ψm than did control cells. Since both Ψm and chemotaxis are regulated by intracellular signaling, we investigated the effects of THC on the activation of multiple signaling pathways. Serum exposure activated several signaling events of which phosphorylation of IκB-α and JNK were regulated in a CB2R-and THC-dependent manner. We conclude that airway epithelial cells are sensitive to both CB2R-dependent and independent effects mediated by THC.
Tetrahydrocannabinol (THC); Cannabinoid receptor; Chemotaxis; Mitochondria; Lentiviral vector; MAPK signaling
Although antiretroviral therapy has the ability to fully restore a normal CD4+ cell count (>500 cells/mm3) in most patients, it is not yet clear whether all patients can achieve normalization of their CD4+ cell count, in part because no study has followed up patients for >7 years.
Three hundred sixty-six patients from 5 clinical cohorts who maintained a plasma human immunodeficiency virus (HIV) RNA level ≤1000 copies/mL for at least 4 years after initiation of antiretroviral therapy were included. Changes in CD4+ cell count were evaluated using mixed-effects modeling, spline-smoothing regression, and Kaplan-Meier techniques.
The majority (83%) of the patients were men. The median CD4+ cell count at the time of therapy initiation was 201 cells/mm3 (interquartile range, 72−344 cells/mm3), and the median age was 47 years. The median follow-up period was 7.5 years (interquartile range, 5.5−9.7 years). CD4+ cell counts continued to increase throughout the follow-up period, albeit slowly after year 4. Although almost all patients (95%) who started therapy with a CD4+ cell count ≥300 cells/mm3 were able to attain a CD4+ cell count ≥500 cells/mm3, 44% of patients who started therapy with a CD4+ cell count <100 cells/mm3 and 25% of patients who started therapy with a CD4+ cell count of 100−200 cells/mm3 were unable to achieve a CD4+ cell count >500 cells/mm3 over a mean duration of follow-up of 7.5 years; many did not reach this threshold by year 10. Twenty-four percent of individuals with a CD4+ cell count <500 cells/mm3 at year 4 had evidence of a CD4+ cell count plateau after year 4. The frequency of detectable viremia (“blips”) after year 4 was not associated with the magnitude of the CD4+ cell count change.
A substantial proportion of patients who delay therapy until their CD4+ cell count decreases to <200 cells/mm3 do not achieve a normal CD4+ cell count, even after a decade of otherwise effective antiretroviral therapy. Although the majority of patients have evidence of slow increases in their CD4+ cell count over time, many do not. These individuals may have an elevated risk of non–AIDS-related morbidity and mortality.
It has been over 25 years since the first diagnosis of what would be known as AIDS. Although great strides in anti-HIV therapeutics have been made, there is still a great need for antiretrovirals that are effective against drug-resistant HIV. Enfuvirtide (ENF) is the first of a new class of fusion inhibitors to be approved by the US Food and Drug Administration for use in combination with other antiretroviral agents among HIV-1 infected patients with previous treatment experience. The inclusion of enfuvirtide in an optimized antiretroviral background regimen for the treatment of HIV-1 infected (treatment-experienced) patients followed the success of two critical clinical trials (TORO: T20 vs Optimized Regimen Only I and II). Even though injection-site reactions persisted in these trials, improved virological and immunological responses were observed among patients. Challenges associated with ENF treatment include the high cost of the drug, injection-site reactions, determining the optimal time to initiate treatment, and the potential for the selection of drug resistant mutants and viral evolution. ENF is a promising novel treatment for HIV infected individuals whose choices for effective treatment are limited by previous treatment and resistance. Understanding the implications of viral fitness and evolution in the presence of ENF treatment is crucial in determining effective and safe treatment regimens, particularly among treatment-experienced patients.
enfuvirtide; HIV; salvage therapy; drug resistance; gp41; evolution
Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.
Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8+ T-lymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.
Investigation of human immunodeficiency virus type 1 (HIV-1) in the genital tract of women is crucial to the development of vaccines and therapies. Previous analyses of HIV-1 in various anatomic sites have documented compartmentalization, with viral sequences from each location that were distinct yet phylogenetically related. Full-length RNA genomes derived from different compartments in the same individual, however, have not yet been studied. Furthermore, although there is evidence that intrapatient recombination may occur frequently, recombinants comprising viruses from different sites within one individual have rarely been documented. We compared full-length HIV-1 RNA sequences in the plasma and female genital tract, focusing on a woman with high HIV-1 RNA loads in each compartment who had been infected heterosexually and then transmitted HIV-1 by the same route. We cloned and sequenced 10 full-length HIV-1 RNA genomes from her genital tract and 10 from her plasma. We also compared viral genomes from the genital tract and plasma of four additional heterosexually infected women, sequencing 164 env and gag clones obtained from the two sites. Four of five women, including the one whose complete viral sequences were determined, displayed compartmentalized HIV-1 genomes. Analyses of full-length, compartmentalized sequences made it possible to document complex intrapatient HIV-1 recombinants that were composed of alternating viral sequences characteristic of each site. These findings demonstrate that the genital tract and blood harbor genetically distinct populations of replicating HIV-1 and provide evidence that recombination between strains from the two compartments contributes to rapid evolution of viral sequence variation in infected individuals.
There is substantial evidence for ongoing replication and evolution of human immunodeficiency virus type 1 (HIV-1), even in individuals receiving highly active antiretroviral therapy. Viral evolution in the presence of antiviral therapy needs to be considered when developing new therapeutic strategies. Phylogenetic analyses of HIV-1 sequences can be used for this purpose but may give rise to misleading results if rates of intrapatient evolution differ significantly. To improve analyses of HIV-1 evolution relevant to studies of pathogenesis and treatment, we developed a Bayesian hierarchical model that incorporates all available sequence data while simultaneously allowing the phylogenetic parameters of each patient to vary. We used this method to examine evolutionary changes in HIV-1 coreceptor usage in response to treatment. We examined patients whose viral populations exhibited a shift in coreceptor utilization in response to therapy. CXCR4 (X4) strains emerged in each patient but were suppressed following initiation of new antiretroviral regimens, so that CCR5-utilizing (R5) strains predominated. By phylogenetically reconstructing the evolutionary relationship of HIV-1 obtained longitudinally from each patient, it was possible to examine the origin of the reemergent R5 virus. Using our Bayesian hierarchical approach, we found that the reemergent R5 virus detectable after therapy was more closely related to the predecessor R5 virus than to the X4 strains. The Bayesian hierarchical approach, unlike more traditional methods, makes it possible to evaluate competing hypotheses across patients. This model is not limited to analyses of HIV-1 but can be used to elucidate evolutionary processes for other organisms as well.
The inhibition of multilineage hematopoiesis which occurs in the severe combined immunodeficiency mouse with transplanted human fetal thymus and liver tissues (SCID-hu Thy/Liv) due to human immunodeficiency virus type 1 (HIV-1) infection is also accompanied by a severe loss of c-Mpl expression on these progenitor cells. Inhibition of colony-forming activity (CFA) of the CD34+ progenitor cells is partially revived to about 40% of mock-infected Thy/Liv implants, following reconstitution of the CD34+ cells that were exposed to HIV-1 infection, in a new Thy/Liv stromal microenvironment of irradiated secondary SCID-hu recipients at 3 weeks post-re-engraftment. In addition, in these reconstituted animals, the proportion of c-Mpl+ CD34+ cells relative to c-Mpl− CD34+ cells increased by about 25%, to 35% of mock-infected implants, suggesting a reacquirement of c-Mpl phenotype by the c-Mpl− CD34+ cells. These results suggest a correlation between c-Mpl expression and multilineage CFA of the human CD34+ progenitor cells that have experienced the effects of HIV-1 infection. Treatment of the secondary-recipient animals with the c-Mpl ligand, thrombopoietin (Tpo), further increased c-Mpl expression and CFA of re-engrafted CD34+ cells previously exposed to virus in the primary implants to about 50 to 70% over that of those re-engrafted CD34+ cells derived from implants of untreated animals. Blocking of c-Mpl with anti-c-Mpl monoclonal antibody in vivo by injecting the SCID-hu animals resulted in the reduction or loss of CFA. Thus, inhibition, absence, or loss of c-Mpl expression as in the c-Mpl− CD34+ subset of cells is the likely cause of CFA inhibition. Further, CFA of the CD34+ cells segregates with their c-Mpl expression. Therefore, c-Mpl may play a role in hematopoietic inhibition during HIV-1 infection, and control of its expression levels may aid in hematopoietic recovery and thereby reduce the incidence of cytopenias occurring in infected individuals.