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ABSTRACT: Antiretroviral therapy (ART) significantly reduced the CD8+ cell non-cytotoxic anti-HIVresponse (CNAR) in twelve HIV-1-infected subjects (p < 0.0001). In separate experiments, CD8+ cells from long term survivors (LTS) were co-cultured with HIV-infected CD4+ cells using varying concentrations of anti-HIV drugs.Theantiviral function of CD8+ cells from four of fourteen LTS was reduced with exposure to 10μM nevirapine(p< 0.05). The antiviral activity of CD8+ cells from two LTS was inhibited by 5μM zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8+ cells and thus could influence the effectiveness of ART.
JAIDS Journal of Acquired Immune Deficiency Syndromes 02/2013; · 4.43 Impact Factor
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ABSTRACT: Objective: In HIV-1 infection, plasmacytoid dendritic cell numbers and function are decreased. No detailed comparisons of PDC responses to various stimuli in HIV-1 infected patients are available. Methods: Using for the first time purified PDC, we compared PDC responses (IFN-α production/cell) to various stimuli in a large number (n=48) of HIV-1 infected patients and healthy volunteers (n=19). TLR7 and TLR9 induced expression of PDC surface activation and maturation markers was also compared in the two populations. Results: We have confirmed that PDC number coincides with CD4+ T cell counts and clinical state. Notably, we have shown that a direct association of PDC function in terms of IFN-α production/cell exists with PDC numbers and CD4+ cell counts when PDC are exposed to a TLR9 ligand and HIV-infected cells, but not with a TLR7 ligand. Moreover, in the HIV-infected subjects but not the healthy controls, the magnitude of IFN-α release per PDC in response to the TLR7 ligand is significantly (p<0.01) lower than that to the TLR9 ligand. However, in both study populations, the TLR7 stimulation in comparison to TLR9 stimulation induced higher expression of PDC surface activation and maturation markers and significantly (p<0.05) decreased the expression of BDCA-2, a negative regulator of interferon. Further, the cross-ligation of BDCA-2 significantly (p<0.05) inhibited TLR9- but not TLR7- induced IFN-α production by PDC from both clinical groups. Conclusions: These findings suggest that differences exist in TLR7- and TLR9- induced IFN-α production by PDC in HIV-infected individuals which are not directly related to BDCA-2 down-modulation.
AIDS research and human retroviruses 11/2012; · 2.18 Impact Factor
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ABSTRACT: While human immunodeficiency virus (HIV) transmission through the adult oral route is rare, mother-to-child transmission (MTCT) through the neonatal/infant oral and/or gastrointestinal route is common. To study the mechanisms of cell-free and cell-associated HIV transmission across adult oral and neonatal/infant oral/intestinal epithelia, we established ex vivo organ tissue model systems of adult and fetal origin. Given the similarity of neonatal and fetal oral epithelia with respect to epithelial stratification and density of HIV-susceptible immune cells, we used fetal oral the epithelium as a model for neonatal/infant oral epithelium. We found that cell-free HIV traversed fetal oral and intestinal epithelia and infected HIV-susceptible CD4(+) T lymphocytes, Langerhans/dendritic cells, and macrophages. To study the penetration of cell-associated virus into fetal oral and intestinal epithelia, HIV-infected macrophages and lymphocytes were added to the surfaces of fetal oral and intestinal epithelia. HIV-infected macrophages, but not lymphocytes, transmigrated across fetal oral epithelia. HIV-infected macrophages and, to a lesser extent, lymphocytes transmigrated across fetal intestinal epithelia. In contrast to the fetal oral/intestinal epithelia, cell-free HIV transmigration through adult oral epithelia was inefficient and virions did not infect intraepithelial and subepithelial HIV-susceptible cells. In addition, HIV-infected macrophages and lymphocytes did not transmigrate through intact adult oral epithelia. Transmigration of cell-free and cell-associated HIV across the fetal oral/intestinal mucosal epithelium may serve as an initial mechanism for HIV MTCT.
Journal of Virology 12/2011; 86(5):2556-70. · 5.40 Impact Factor
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ABSTRACT: Acquired immune deficiency syndrome (AIDS) was first described 30 years ago in a report from the US Centers for Disease Control. Two years later the causative virus was identified and afterwards named the human immunodeficiency virus (HIV). This article reviews the progress made in the three decades since the recognition of AIDS and the discovery of HIV, with respect to the virus, the infected cell, and the host, as well as directions for future studies.
European Journal of Immunology 12/2011; 41(12):3401-11. · 5.10 Impact Factor
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ABSTRACT: Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by > 95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.
Journal of immunological methods 07/2011; 372(1-2):22-9. · 2.35 Impact Factor
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Konstance Knox,
Donald Carrigan,
Graham Simmons,
Fernando Teque,
Yanchen Zhou,
John Hackett,
Xiaoxing Qiu,
Ka-Cheung Luk,
Gerald Schochetman,
Allyn Knox,
Andreas M Kogelnik, Jay A Levy
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ABSTRACT: Members of the gammaretroviruses--such as murine leukemia viruses (MLVs), most notably XMRV [xenotropic murine leukemia virus (X-MLV)-related virus--have been reported to be present in the blood of patients with chronic fatigue syndrome (CFS). We evaluated blood samples from 61 patients with CFS from a single clinical practice, 43 of whom had previously been identified as XMRV-positive. Our analysis included polymerase chain reaction and reverse transcription polymerase chain reaction procedures for detection of viral nucleic acids and assays for detection of infectious virus and virus-specific antibodies. We found no evidence of XMRV or other MLVs in these blood samples. In addition, we found that these gammaretroviruses were strongly (X-MLV) or partially (XMRV) susceptible to inactivation by sera from CFS patients and healthy controls, which suggested that establishment of a successful MLV infection in humans would be unlikely. Consistent with previous reports, we detected MLV sequences in commercial laboratory reagents. Our results indicate that previous evidence linking XMRV and MLVs to CFS is likely attributable to laboratory contamination.
Science 06/2011; 333(6038):94-7. · 31.20 Impact Factor
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ABSTRACT: HIV replication is suppressed in vitro by a CD8(+) cell noncytotoxic antiviral response (CNAR). This activity directly correlates with an asymptomatic clinical state. The objective of this study was to identify the phenotype of CD8(+) cell subsets having strong CNAR activity. CD8(+) cell subset frequencies and CNAR levels were measured for human immunodeficiency virus (HIV)-uninfected individuals and three groups of HIV type 1 (HIV-1)-infected individuals: asymptomatic individuals with low-level viremia (vHIV), antiretroviral-drug-treated subjects with undetectable virus levels (TxHIV), and therapy-naïve aviremic elite controllers (EC). CD8(+) cells from the vHIV individuals exhibited the highest HIV-suppressing activity and had elevated frequencies of CD45RA(-) CD27(+) and PD-1(+) (CD279(+)) cells. Functional assessments of CD8(+) cells sorted into distinct subsets established that maximal CNAR activity was mediated by CD45RA(-) CCR7(-) CD27(+) and PD-1(+) CD8(+) cells. T cell receptor (TCR) repertoire profiles of CD8(+) cell subsets having strong CNAR activity exhibited increased perturbations in comparison to those of inactive subsets. Together, these studies suggest that CNAR is driven by HIV replication and that this antiviral activity is associated with oligoclonally expanded activated CD8(+) cells expressing PD-1 and having a transitional memory cell phenotype. The findings better describe the identity of CD8(+) cells showing CNAR and should facilitate the evaluation of this important immune response in studies of HIV pathogenesis, resistance to infection, and vaccine development.
Journal of Virology 02/2011; 85(4):1696-705. · 5.40 Impact Factor
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Florencia Pereyra,
Xiaoming Jia,
Paul J McLaren,
Amalio Telenti,
Paul I W de Bakker,
Bruce D Walker,
Stephan Ripke,
Chanson J Brumme,
Sara L Pulit,
Mary Carrington, [......],
Alan Winston,
Clifford G Wlodaver,
Angelique van't Wout,
David P Wright,
Otto O Yang,
David L Yurdin,
Brandon W Zabukovic,
Kimon C Zachary,
Beth Zeeman,
Meng Zhao
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ABSTRACT: Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.
Science 12/2010; 330(6010):1551-7. · 31.20 Impact Factor
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ABSTRACT: Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells - and not those that passed through the adult cells - remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission.
Virology 11/2010; 409(2):211-22. · 3.35 Impact Factor
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Lishomwa C Ndhlovu,
Elizabeth Sinclair,
Lorrie Epling,
Qi Xuan Tan,
Terence Ho,
Aashish R Jha,
Ijeoma Eccles-James,
Camilla Tincati, Jay A Levy,
Douglas F Nixon,
Frederick M Hecht,
Jason D Barbour
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ABSTRACT: Little is known about the manipulation of IL-17 producing CD4+ T cells (T(H)17) on a per-cell basis in humans in vivo. Previous studies on the effects of IL-2 on IL-17 secretion in non-HIV models have shown divergent results. We hypothesized that IL-2 would mediate changes in IL-17 levels among recently HIV-1-infected adults receiving anti-retroviral therapy. We measured cytokine T cell responses to CD3/CD28, HIV-1 Gag, and CMV pp65 stimulation, and changes in multiple CD4+ T cell subsets. Those who received IL-2 showed a robust expansion of naive and total CD4+ T cell counts and T-reg counts. However, after IL-2 treatment, the frequency of T(H)17 cells declined, while counts of T(H)17 cells did not change due to an expansion of the CD4+ naïve T cell population (CD27+CD45RA+). Counts of HIV-1 Gag-specific T cells declined modestly, but CMV pp65 and CD3/CD28 stimulated populations did not change. Hence, in contrast with recent studies, our results suggest IL-2 is not a potent in vivo regulator of T(H)17 cell populations in HIV-1 disease. However, IL-2-mediated T-reg expansions may selectively reduce responses to certain antigen-specific populations, such as HIV-1 Gag.
Journal of Clinical Immunology 09/2010; 30(5):681-92. · 3.08 Impact Factor
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Jay A Levy
AIDS (London, England) 02/2009; 23(2):147-60. · 4.91 Impact Factor
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ABSTRACT: The relationship of elevated T cell activation to altered T cell differentiation profiles, each defining features of HIV-1 infection, has not been extensively explored. We hypothesized that anti-retroviral suppression of T cell activation levels would lead to alterations in the T cell differentiation of total and HIV-1 specific CD8+ T cell responses among recently HIV-1 infected adults.
We performed a longitudinal study simultaneously measuring T cell activation and maturation markers on both total and antigen-specific T cells in recently infected adults: prior to treatment; after the initiation of HAART; and after treatment was halted. Prior to treatment, HIV-1 Gag-specific CD8+ T cells were predominantly of a highly activated, intermediate memory (CD27+CD28-) phenotype, while CMV pp65-specific CD8+ T cells showed a late memory (CD27-CD28-), low activation phenotype. Participants with the highest fraction of late memory (CD27-CD28-) HIV-1-specific CD8+ T cells had higher CD4+ T cell counts (rho = +0.74, p = 0.004). In turn, those with the highest fraction of intermediate memory (CD27+ CD28-) HIV-1 specific CD8+ T cells had high total CD8+ T cell activation (rho = +0.68, p = 0.01), indicating poorer long-term clinical outcomes. The HIV-1 specific T cell differentiation profile was not readily altered by suppression of T cell activation following HAART treatment.
A more differentiated, less activated HIV-1 specific CD8+ T cell response may be clinically protective. Anti-retroviral treatment initiated two to four months after infection lowered T cell activation but had no effect on the differentiation profile of the HIV-1-specific response. Intervention during the first month of acute infection may be required to shift the differentiation phenotype of HIV-1 specific responses to a more clinically favorable profile.
PLoS ONE 02/2009; 4(2):e4408. · 4.09 Impact Factor
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ABSTRACT: CD8(+) cells from healthy, asymptomatic HIV-1-infected individuals can inhibit HIV-1 replication in naturally or acutely infected CD4(+) cells in the absence of cell killing. This CD8(+) cell noncytotoxic anti-HIV response (CNAR) is mediated by a soluble CD8(+) cell antiviral factor (CAF). CNAR/CAF inhibits HIV-1 replication by blocking viral RNA transcription. HIV transcription is regulated by a variety of cis-acting DNA sequence elements within the proviral long terminal repeat (LTR). We hypothesized that one of the HIV-1 LTR proviral DNA sequence elements that binds host cell transcriptional factors is involved in this antiviral activity. To assess this possibility, we constructed full-length infectious HIV-1 molecular clones with mutations in the LTR elements NFAT, AP-1, IL-2 homology region, and the downstream ISRE. We also tested full-length infectious molecular clones that had deletions of either the NF-kappaB or Sp1 sites of the LTR or lacked functional Tat and TAR elements. Viruses generated from these molecular clones were used to acutely infect CD4(+) cells that subsequently were either co-cultured with CD8(+) cells from individuals that exhibited strong CNAR or cultured with CAF-containing fluids. The replication of all of the mutant HIV-1 viruses tested was substantially reduced in the presence of CNAR/CAF. These findings suggest that other regions in the viral LTR or other host cell processes are involved in the transcriptional block elicited by CNAR/CAF.
Virology 04/2008; 373(1):30-8. · 3.35 Impact Factor
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ABSTRACT: We have developed a single-step reverse transcriptase kinetic PCR assay (kRT-PCR) to accurately determine the expression of each of the 24 TCRbetaV gene families in CD8(+) cells. We analyzed the long-term effects of highly active antiretroviral therapy (HAART) on the stability of the CD8(+) T cell receptor (TCR) repertoire in a cohort of 15 treated and 10 untreated individuals diagnosed with human immunodeficiency virus (HIV) infection. The CD4(+) TCR repertoire was studied in a second cohort receiving interleukin-2 infusions in addition to HAART. Analysis was based on kinetic (quantitative) reverse-transcription PCR (kRT-PCR) of the TCR variable B gene (TCRbetaV). Expression of each of the 24 Vbeta families was assessed at baseline immediately after infection and following initiation of HAART at 2, 4, 12, 24, and up to 192 weeks in 24-week intervals. Statistically significant family-specific expression changes were observed between treated and untreated individuals for 10 TCRbetaV families. Overall, when compared to untreated patients, a more stable expression of TCR genes was observed for HAART-treated individuals. Interestingly, this difference did not correlate with either CD4 or CD8 counts, which follow the expected curves for treated and untreated patients. When we applied our quantitative analysis to IL-2-treated patients we observed a rapid polyclonal activation of the repertoire. These results suggest that homeostasis in the T cell receptor repertoire is more robust in those patients who stay on HAART for a long time and confirm the polyclonal stimulating capacity of IL-2.
AIDS Research and Human Retroviruses 06/2007; 23(5):741-7. · 2.25 Impact Factor
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ABSTRACT: CD8(+) cells from healthy HIV-1-infected individuals suppress human immunodeficiency virus (HIV) replication in infected cells by a non-cytotoxic mechanism. This activity is associated with the production of a soluble CD8(+) cell antiviral factor (CAF) that inhibits viral replication at the level of transcription. Strong CD8(+) cell non-cytotoxic anti-HIV responses (CNARs) correlate with an asymptomatic state and long-term survival of HIV-infected individuals. This antiviral activity is lost when the infected individual advances to disease. In attempts to define the gene(s) mediating CNAR we have evaluated differential gene expression between CD8(+) cells from infected subjects with high CNAR and CD8(+) cells from uninfected controls that lack this activity. The expression analysis, using the Affymetrix GeneChip Human Genome U133 Set, indicated that 18% of the genes were differentially expressed (DE) of which 9.2% were up-regulated. A total of 568 genes were up-regulated with a >2.0-fold difference in expression levels and a >50% concordance of difference call. Stringent selection criteria narrowed down the list to 52 up-regulated 'high confidence genes' (> or = 75% concordance). These genes function in a wide variety of cellular processes and include 13 associated with immunologic activity.
Virology 05/2007; 362(1):217-25. · 3.35 Impact Factor
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PLoS Pathogens 05/2007; 3(4):e43. · 9.13 Impact Factor
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ABSTRACT: HIV-specific antibodies become detectable and continue to increase in frequency during primary infection. The effects of early antiretroviral treatment (ART) and its discontinuation on the evolution of this immune response have not been systematically analyzed. To investigate the associations between antibody titer, viral load, and ART, we used a less-sensitive enzyme-linked immunosorbant assay (LS-EIA) to measure changes in HIV-1-specific antibody levels in treated and untreated subjects undergoing primary infection. In this longitudinal study, antibody levels gradually increased in therapy-naive subjects, reaching a plateau approximately 40 weeks postinfection. In contrast, antibody titers remained low among subjects receiving ART. Subjects who discontinued ART exhibited a more rapid rise in antibody titers than therapy-naive subjects, suggesting the presence of an enhanced B cell response. These results demonstrate that early ART prevents the typical evolution of the HIV-1-specific antibody response and can alter the expected kinetics of this response in subjects discontinuing therapy.
AIDS Research and Human Retroviruses 08/2006; 22(7):640-7. · 2.25 Impact Factor
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ABSTRACT: Reduced dendritic cell (DC) frequencies and functions in individuals with longstanding HIV-1 infection are predictive of opportunistic infections and AIDS. To investigate possible early alterations in DC levels after HIV infection, we prospectively examined plasmacytoid dendritic cell (pDC) and myeloid dendritic cell (mDC) frequencies and plasma IFN-alpha levels in patients undergoing primary HIV-1 infection (PHI).
Peripheral blood DC frequencies and absolute counts were determined by flow cytometry. Plasma IFN-alpha levels were measured by enzyme-linked immunosorbent assay (ELISA).
In comparison to uninfected subjects, pDC, but not mDC, levels were reduced (P < 0.001) in subjects with PHI, especially in those with high viral loads or low CD4 T-cell counts. During 24-48 weeks of observation, untreated subjects experienced slight declines in pDC and CD4 T-cell levels. In contrast, subjects initiating early antiretroviral therapy (ART) exhibited increases (P < 0.001) in pDC and CD4 T-cell counts. No effect of treatment on mDC counts was observed. Circulating plasma IFN-alpha was undetectable by ELISA regardless of the duration of HIV-1 infection.
PHI is characterized by a reduction in pDC and CD4 T-cell counts that correlates with the magnitude of virus replication and is not evidenced by the mDC count or plasma IFN-alpha level. Early ART appears to have similar restorative effects on pDC and CD4 T-cell counts.
AIDS 07/2006; 20(9):1247-52. · 6.24 Impact Factor
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C Bradley Hare,
Brandee L Pappalardo,
Michael P Busch,
Annika C Karlsson,
Bruce H Phelps,
Steven S Alexander,
Christopher Bentsen,
Clarissa A Ramstead,
Douglas F Nixon, Jay A Levy,
Frederick M Hecht
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ABSTRACT: We assessed human immunodeficiency virus (HIV) antibody seroreversion among individuals initiating antiretroviral therapy (ART) during acute/early HIV infection and determined whether seroreversion was associated with loss of cytotoxic T lymphocyte responses.
Subjects in a cohort with acute/early HIV infection (<12 months into infection) who initiated ART within 28 days after study entry and maintained HIV type 1 ribonucleic acid levels of < or =500 copies/mL for >24 weeks were selected. Two clinically available second-generation enzyme immunoassays (EIAs) and a confirmatory Western blot were used to screen subjects for antibody reversion. Those with negative screening test results underwent additional antibody testing, including a third-generation EIA, and were assessed for cytotoxic T lymphocyte responses.
Of 87 subjects identified, 12 (14%) had negative antibody test results at the start of ART; all 12 had seroconversion, although 1 had seroconversion only on a third-generation EIA. Of the 87 subjects, 6 (7%) had seroreversion on at least 1 EIA antibody assay while receiving ART during a median follow-up of 90 weeks. The only clinical predictor of seroreversion was a low baseline "detuned" (less sensitive) antibody. Cytotoxic T lymphocyte responses to HIV Gag peptides were detected in 4 of 5 subjects with seroreversion who could be tested. All 5 who had seroreversion who stopped ART experienced virologic rebound and antibody evolution.
HIV antibody seroconversion on second-generation EIA antibody tests may fail to occur when ART is initiated early. Seroreversion was not uncommon among subjects treated early, although cytotoxic T lymphocyte responses to HIV antigens remained detectable in most subjects. Antibody seroreversion did not indicate viral eradication. A third-generation EIA was the most sensitive test for HIV antibodies.
Clinical Infectious Diseases 04/2006; 42(5):700-8. · 9.15 Impact Factor
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ABSTRACT: Plasmacytoid and myeloid dendritic cells are reduced in AIDS patients. The number of these circulating cells was assessed cross-sectionally and longitudinally in 27 uninfected and 72 HIV-infected subjects on and off antiretroviral therapy. The plasmacytoid dendritic cell numbers were significantly reduced in the HIV-infected subjects compared to controls (p < 0.001). This reduction correlated directly with CD4+ cell counts (p < 0.001) and inversely with viral load (p < 0.001). These associations were found to a lesser degree for the myeloid dendritic cells. Intra-assay variability of these dendritic cell counts was < 10%. Antiretroviral therapy significantly increased plasmacytoid dendritic cell (p < 0.001) and CD4+ cell (p = 0.05) counts at 8 months by 76.9% and 19%, respectively. The plasmacytoid dendritic cell levels responded more readily to viral load increases and decreases than CD4+ cells. Circulating plasmacytoid dendritic cells may provide important additional information about immune function in HIV-infected subjects receiving or not receiving antiretroviral therapy.
Journal of Clinical Immunology 02/2006; 26(1):55-64. · 3.08 Impact Factor
