[Show abstract][Hide abstract] ABSTRACT: HIV-1-specific T-cell responses in exposed seronegative subjects suggest that a viral breach of the exposure site is more common than current transmission rates would suggest and that host immunity can extinguish subsequent infection foci. The Preexposure Prophylaxis Initiative (iPrEx) chemoprophylaxis trial provided an opportunity to rigorously investigate these responses in a case-control immunology study; 84 preinfection peripheral blood mononuclear cell samples from individuals enrolled in the iPrEx trial who later seroconverted were matched with 480 samples from enrolled subjects who remained seronegative from both the placebo and active treatment arms. T-cell responses to HIV-1 Gag, Protease, Integrase, Reverse Transcriptase, Vif, and Nef antigens were quantified for all subjects in an IFN-γ enzyme-linked immunospot (ELISpot) assay. IFN-γ responses varied in magnitude and frequency across subjects. A positive response was more prevalent in those who remained persistently HIV-1-negative for Gag (P = 0.007), Integrase (P < 0.001), Vif (P < 0.001), and Nef (P < 0.001). When correlated with outcomes in the iPrEx trial, Vif- and Integrase-specific T-cell responses were associated with reduced HIV-1 infection risk [hazard ratio (HR) = 0.36, 95% confidence interval (95% CI) = 0.19-0.66 and HR = 0.52, 95% CI = 0.28-0.96, respectively]. Antigen-specific responses were independent of emtricitabine/tenofovir disoproxil fumarate use. IFN-γ secretion in the ELISpot was confirmed using multiparametric flow cytometry and largely attributed to effector memory CD4+ or CD8+ T cells. Our results show that HIV-1-specific T-cell immunity can be detected in exposed but uninfected individuals and that these T-cell responses can differentiate individuals according to infection outcomes.
[Show abstract][Hide abstract] ABSTRACT: DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double-stranded DNA sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC-mediated plasmid degradation. We showed that plasmid sensing via STING and TBK-1 leads to IFN-β induction, which results in APOBEC3A mRNA upregulation through a mechanism involving PKC signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to 10-fold reduction in intracellular plasmid levels and plasmid-encoded mRNA, and 2.6-fold reduction in GFP-expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2-specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN-γ-secreting T cells. Our study brings new insights into the intracellular machinery involved in double-stranded DNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.Immunology and Cell Biology accepted article preview online, 08 May 2015. doi:10.1038/icb.2015.53.
Immunology and Cell Biology 05/2015; DOI:10.1038/icb.2015.53 · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Psoriasis patients have relatively infrequent cutaneous viral infections compared to atopic dermatitis patients. Increased expression of four antiviral proteins (MX1, BST2, ISG15 and OAS2) has been reported in psoriatic skin and genetic studies of psoriasis have identified susceptibility genes in antiviral pathways.Objective
To determine if psoriasis is associated with pervasive expression of antiviral genes in skin and blood.Methods
We performed RNA sequencing on skin samples of 18 subjects with chronic plaque psoriasis and 16 healthy controls. We examined the expression of a predefined set of 42 antiviral genes, each of which has been shown in previous studies to inhibit viral replication. In parallel, we examined antiviral gene expression in atopic dermatitis, non-lesional psoriatic skin and psoriatic blood. We performed HIV-1 infectivity assays in CD4+ peripheral blood T cells from psoriatic and healthy individuals.ResultsWe observed significant overexpression of 16 antiviral genes in lesional psoriatic skin, with a greater than two-fold increase in ISG15, RSAD2, IRF7, MX2 and TRIM22 (P < 1E-07). None of these genes was overexpressed in atopic dermatitis skin (P < 0.0001) or non-lesional psoriatic skin. In contrast to the skin compartment, no differences in antiviral gene expression were detected in the peripheral blood of psoriasis cases compared to healthy controls. CD4+ T cells from both psoriatic and healthy patients supported HIV-1 infection at a similar rate.Conclusion
Our findings highlight psoriasis as an inflammatory disease with cutaneous but not systemic immune activation against viral pathogens.
Journal of the European Academy of Dermatology and Venereology 03/2015; 29(10). DOI:10.1111/jdv.13091 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we investigated the expression levels of host restriction factors in six untreated HIV-1-positive patients
over the course of infection. We found that the host restriction factor gene expression profile consistently increased over
time and was significantly associated with CD4+ T cell activation and viral load. Our data are among the first to demonstrate the dynamic nature of host restriction factors
in vivo over time.
Journal of Virology 07/2014; 88(19). DOI:10.1128/JVI.01771-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several host-encoded antiviral factors suppress HIV-1 replication in a cell-autonomous fashion in vitro. The relevance of these defenses to the control of HIV-1 in vivo remains to be elucidated. We hypothesized that cellular restriction of HIV-1 replication plays a significant role in the observed suppression of HIV-1 in "elite controllers", individuals who maintain undetectable levels of viremia in the absence of antiretroviral therapy (ART). We comprehensively compared the expression levels of 34 host restriction factors and cellular activation levels in CD4+ T cells and sorted T cell subsets between elite controllers, HIV-1-infected (untreated) non-controllers, ART-suppressed, and uninfected individuals.
Expression of schlafen 11, a codon usage-based inhibitor of HIV-1 protein synthesis, was significantly elevated in CD4+ T cells from elite controllers as compared to both non-controllers (p=0.048) and ART-suppressed individuals (p=0.024), with this effect most apparent in central memory CD4+ T cells. Schlafen 11 expression levels were comparable between controllers and uninfected individuals. Cumulative restriction factor expression was positively correlated with CD4+ T cell activation (r2=0.597, p<0.0001), viral load (r2=0.34, p=0.015), and expression of ISG15 (r2=0.73, p<0.0001), a marker of interferon exposure. APOBEC3C, APOBEC3D, CTR9, TRIM26, and TRIM32 were elevated in controllers with respect to ART-suppressed individuals, while levels were comparable to uninfected individuals and non-controllers.
Host restriction factor expression typically scales with cellular activation levels. However, the elevated mRNA and protein expression of schlafen 11, despite low activation and viral load, violates the global pattern and may be a signature characteristic of HIV-1 elite control.
[Show abstract][Hide abstract] ABSTRACT: Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome, but are suppressed by host-factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, while reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif- and Vpr-dependent manner. In primary CD4(+) cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extra-chromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity.
Journal of Virology 10/2013; 87(24). DOI:10.1128/JVI.02257-13 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression of cell-intrinsic antiviral factors suppresses HIV-1 replication. We hypothesized that cellular activation modulates host restriction and susceptibility to HIV-1 infection. We measured the gene expression of 34 antiviral factors in healthy PBMC. Cellular activation induced expression of ISG15, TRIM5α, BST-2/tetherin and certain APOBEC3-family members. Expression of RTF1, PAF1, TRIM11, TRIM26 and BST-2/tetherin correlated with decreased HIV-1 infectivity. This study demonstrates synchronous effects of activation-induced antiviral genes on HIV-1 infectivity, providing candidates for pharmacological manipulation.
Journal of Virology 08/2013; 87(21). DOI:10.1128/JVI.02128-13 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genetic background of HIV-1-infected subjects, particularly the HLA class I haplotype, appears to be critical in determining disease progression rates, thought to be a result of the role of HIV-1-specific CD8(+) T cell responses. The HLA-B*57 allele is strongly associated with viremic suppression and slower disease progression. However, there is considerable heterogeneity in HIV-1 disease progression rates among HLA-B*57-positive subjects, suggesting that additional factors may help to contain viral replication. In this report, we investigated the association between host restriction factors, other established immunological parameters, and HLA type in HIV-1-seronegative individuals. Our results demonstrate that healthy, uninfected HLA-B*57-positive individuals exhibit significantly higher gene-expression levels of host restriction factors, such as APOBEC3A, APOBEC3B, BST-2/tetherin, and ISG15. Interestingly, HLA-B*57 individuals have significantly lower CD4(+) T cell frequencies but harbor slightly more activated CD4(+) T cells compared with their HLA-B*35 counterparts. We detected significant correlations between CD4(+) T cell activation and expression of several APOBEC3 family members, BST-2/tetherin, SAMHD1, and TRIM5α in HLA-B*57-positive individuals. To our knowledge, this is the first report showing distinct associations between host restriction factors and HLA class I genotype. Our results provide insights into natural protection mechanisms and immunity against HIV-1 that fall outside of classical HLA-mediated effects.
[Show abstract][Hide abstract] ABSTRACT: APOBEC3 proteins mediate potent anti-retroviral activity by hypermutating the retroviral genome during reverse transcription. To counteract APOBEC3 and gain a replicative advantage, lentiviruses such as HIV-1 and SIV have evolved the Vif protein, which targets APOBEC3 proteins for proteasomal degradation. However, the proteasome plays a critical role in the generation of T cell peptide epitopes. Whether Vif-mediated destruction of APOBEC3 proteins leads to the generation and presentation of APOBEC3-derived T cell epitopes on the surface of lentivirus-infected cells remains unknown. Here, using peptides derived from multiple Vif-sensitive APOBEC3 proteins, we identified APOBEC3-specific T cell responses in both HIV-1-infected patients and SIV-infected rhesus macaques. These results raise the possibility that these T cell responses may be part of the larger anti-retroviral immune response.
Journal of Virology 03/2013; 87(11). DOI:10.1128/JVI.00579-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1. Immune pressure exerted by ARF CD8(+) T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo.
In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased.
These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.
PLoS ONE 06/2012; 7(6):e39311. DOI:10.1371/journal.pone.0039311 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Upon activation, CD4(+) T cells release cytokines, chemokines, and other soluble factors that influence the kinetics of HIV-1 replication in macrophages (M). In this article, we show that activation of human primary T cells suppresses the early stages of HIV-1 replication in human primary Mφ by downregulating the main cellular receptor for the virus CD4. The secreted factors responsible for this effect have a molecular mass greater than conventional cytokines, are independent of Th1 or Th2 polarization, and are not IFN-γ, IL-16, RANTES, or macrophage inhibitory factor, as revealed by cytokine array analysis and neutralization assays. CD4 downregulation is entirely posttranslational and involves serine phosphorylation of CD4 and its targeting to an intracellular compartment destined for acidification and degradation. CD4 downregulation is dependent on the activities of both protein kinase C and NF-κB as well as the proteasomes. Using high-resolution liquid chromatography-tandem mass spectrometry analysis in conjugation with label-free protein quantitation software, we found that proteins that promote Mφ adherence and spreading, such as attractin, fibronectin, and galectin-3-binding protein, were significantly overrepresented in the activated T cell supernatant fractions. These results reveal the existence of previously unreported anti-HIV-1 proteins, released by activated T cells that downregulate CD4 expression, and are of fundamental importance to understand the kinetics of HIV infection in vivo.
The Journal of Immunology 06/2011; 187(2):748-59. DOI:10.4049/jimmunol.1003678 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human macrophages (Mφ) express low levels of CD4 glycoprotein, which is constitutively recycled, and 40-50% of its localization is intracellular at steady-state. Although CD4-interacting proteins in lymphoid cells are well characterised, little is known about the CD4 protein interaction-network in human Mφ, which notably lack LCK, a Src family protein tyrosine kinase believed to stabilise CD4 at the surface of T cells. As CD4 is the main cellular receptor used by HIV-1, knowledge of its molecular interactions is important for the understanding of viral infection strategies.
We performed large-scale anti-CD4 immunoprecipitations in human primary Mφ followed by high-resolution mass spectrometry analysis to elucidate the protein interaction-network involved in induced CD4 internalization and degradation. Proteomic analysis of CD4 co-immunoisolates in resting Mφ showed CD4 association with a range of proteins found in the cellular cortex, membrane rafts and components of clathrin-adaptor proteins, whereas in induced internalization and degradation CD4 is associated with components of specific signal transduction, transport and the proteasome.
This is the first time that the anti-CD4 co-immunoprecipitation sub-proteome has been analysed in human primary Mφ. Our data have identified important Mφ cell surface CD4-interacting proteins, as well as regulatory proteins involved in internalization and degradation. The data give valuable insights into the molecular pathways involved in the regulation of CD4 expression in Mφ and provide candidates/targets for further biochemical studies.
PLoS ONE 04/2011; 6(4):e18690. DOI:10.1371/journal.pone.0018690 · 3.23 Impact Factor