Shariq Mujib

HERV-K–specific T cells eliminate diverse HIV-1/2 and SIV primary isolates

The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1-infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of CD4+ T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)-specific CD8+ T cells obtained from HIV-1-infected human subjects responded to HIV-1-infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)-specific CD8+ T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)-specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)-targeted HIV-1 vaccines and immunotherapeutics.

Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway.

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8+ and Th1 CD4+ T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3+ cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α. In this study, we examined potential inducers of Tim-3 on primary human T cells. Direct HIV-1 infection of CD4+ T cells, or LPS, found to be elevated in HIV-1 infection, did not induce Tim-3 on T cells. Tim-3 was induced by the common γ-chain (γc) cytokines IL-2, IL-7, IL-15, and IL-21 but not IL-4, in an Ag-independent manner and was upregulated on primary T cells in response to TCR/CD28 costimulation, as well as γc cytokine stimulation with successive divisions. γc cytokine-induced Tim-3 was found on naive, effector, and memory subsets of T cells. Tim-3+ primary T cells were more prone to apoptosis, particularly upon treatment with galectin-9, a Tim-3 ligand, after cytokine withdrawal. The upregulation of Tim-3 could be blocked by the addition of a PI3K inhibitor, LY 294002. Thus, Tim-3 can be induced via TCR/CD28 costimulation and/or γc cytokines, likely through the PI3K pathway.

Tim-3 Negatively Regulates Cytotoxicity in Exhausted CD8+ T Cells in HIV Infection

Cytotoxic CD8+ T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell ‘exhaustion’ is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8+ T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill autologous HIV infected CD4+ target cells. Surprisingly, Tim-3+ CD8+ T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and d) their ability to suppress HIV infection of CD4+ T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8+ T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8+ T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.

Early Initiation of Combination Antiretroviral Therapy in HIV-1–Infected Newborns Can Achieve Sustained Virologic Suppression With Low Frequency of CD4+ T Cells Carrying HIV in Peripheral Blood

BACKGROUND A human immunodeficiency virus type 1 (HIV-1)-infected infant started on combination antiretroviral therapy (cART) at 30 hours of life was recently reported to have no detectable plasma viremia after discontinuing cART. The current study investigated the impact of early cART initiation on measures of HIV-1 reservoir size in HIV-1-infected children with sustained virologic suppression. METHODS Children born to HIV-1-infected mothers and started on cART within 72 hours of birth at 3 Canadian centers were assessed. HIV serology, HIV-1-specific cell-mediated immune responses, plasma viremia, cell-associated HIV-1 DNA and RNA, presence of replication-competent HIV-1, and HLA genotype were determined for HIV-1-infected children with sustained virologic suppression. RESULTS Of 136 cART-treated children, 12 were vertically infected (8.8%). In the 4 who achieved sustained virologic suppression, HIV serology, HIV-1-specific cell-mediated immune responses (Gag, Nef), and ultrasensitive viral load were negative. HIV-1 DNA was not detected in enriched CD4(+) T cells of the 4 children (<2.6 copies/10(6) CD4(+) T cells), whereas HIV-1 RNA was detected (19.5-130 copies/1.5 µg RNA). No virion-associated HIV-1 RNA was detected following mitogenic stimulation of peripheral blood CD4(+) T cells (5.4-8.0 million CD4(+) T cells) in these 4 children, but replication competent virus was detected by quantitative co-culture involving a higher number of cells in 1 of 2 children tested (0.1 infectious units/10(6) CD4(+) T cells). CONCLUSIONS In perinatally HIV-1-infected newborns, initiation of cART within 72 hours of birth may significantly reduce the size of the HIV-1 reservoirs. Cessation of cART may be necessary to determine whether functional HIV cure can be achieved in such children.

T Cell Ig and Mucin Domain–Containing Protein 3 Is Recruited to the Immune Synapse, Disrupts Stable Synapse Formation, and Associates with Receptor Phosphatases

CD8+ CTLs are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g., IFN-γ) to aid this response. However, during cancer and chronic viral infections, such as with HIV, this CTL response is progressively impaired due to a process called T cell exhaustion. Previous work has shown that the glycoprotein T cell Ig and mucin domain–containing protein 3 (Tim-3) plays a functional role in establishing T cell exhaustion. Tim-3 is highly upregulated on virus and tumor Ag-specific CD8+ T cells, and antagonizing Tim-3 helps restore function of CD8+ T cells. However, very little is known of how Tim-3 signals in CTLs. In this study, we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in primary human CD8+ T cells. Tim-3 was found within CD8+ T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted in a significantly greater number of stable synapses being formed between Tim-3hiCD8+ T cells and target cells, suggesting that Tim-3 plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse, and characterizing these interactions could aid in the development of therapeutics to restore Tim-3–mediated immune dysfunction.

LINE-1 Retrotransposable Element DNA Accumulates in HIV-1-Infected Cells

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, whereas 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 extrachromosomal. 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.

Human Immunodeficiency Virus Type 1 Escapes from Interleukin-2-Producing CD4+ T-Cell Responses without High-Frequency Fixation of Mutations

ABSTRACT The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T-cell responses has been associated with the immunological control of HIV-1 replication; however, the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4+ T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells, each of the individuals in the present study exhibited progressive disease, with one individual showing rapid progression. In this rapid progressor, three IL-2-producing HIV-1 Gag-specific CD4+ T-cell responses were identified and mapped to the following optimal epitopes: HIVWASRELER, REPRGSDIAGT, and FRDYVDRFYKT. Responses to these epitopes in peripheral blood mononuclear cells were monitored longitudinally to >1 year postinfection, and contemporaneous circulating plasma viruses were sequenced. A variant of the FRDYVDRFYKT epitope sequence, FRDYVDQFYKT, was observed in 1/21 plasma viruses sequenced at 5 months postinfection and 1/10 viruses at 7 months postinfection. This variant failed to stimulate the corresponding CD4+ T-cell clone and thus constitutes an escape mutant. Responses to each of the three Gag epitopes were rapidly lost, and this loss was accompanied by a loss of antigen-specific cells in the periphery as measured by using an FRDYVDRFYKT-presenting major histocompatibility complex class II tetramer. Highly active antiretroviral therapy was associated with the reemergence of FRDYVDRFYKT-specific cells by tetramer. Thus, our data support that IL-2-producing HIV-1-specific CD4+ T-cell responses can exert immune pressure during early HIV-1 infection but that the inability of these responses to enforce enduring control of viral replication is related to the deletion and/or dysfunction of HIV-1-specific CD4+ T cells rather than to the fixation of escape mutations at high frequencies.

HIV Delays IFN-α Production from Human Plasmacytoid Dendritic Cells and Is Associated with SYK Phosphorylation

Plasmacytoid dendritic cells (pDC) are the major producers of type I interferons (IFNs) in humans and rapidly produce IFN-α in response to virus exposure. Although HIV infection is associated with pDC activation, it is unclear why the innate immune response is unable to effectively control viral replication. We systematically compared the effect of HIV, Influenza, Sendai, and HSV-2 at similar target cell multiplicity of infection (M.O.I.) on human pDC function. We found that Influenza, Sendai, HSV-2 and imiquimod are able to rapidly induce IFN-α production within 4 hours to maximal levels, whereas HIV had a delayed induction that was maximal only after 24 hours. In addition, maximal IFN-α induction by HIV was at least 10 fold less than that of the other viruses in the panel. HIV also induced less TNF-α and MIP-1β but similar levels of IP-10 compared to other viruses, which was also mirrored by delayed upregulation of pDC activation markers CD83 and CD86. BDCA-2 has been identified as an inhibitory receptor on pDC, signaling through a pathway that involves SYK phosphorylation. We find that compared to Influenza, HIV induces the activation of the SYK pathway. Thus, HIV delays pDC IFN-α production and pDC activation via SYK phosphorylation, allowing establishment of viral populations.

Human endogenous retrovirus K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected in HIV-1-infected subjects using standard peptide matrix-based screening

ABSTRACT T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15mer peptide mapping.

Self-assembling peptide for co-delivery of HIV-1 CD8+ T cells epitope and Toll-like receptor 7/8 agonists R848 to induce maturation of monocyte derived dendritic cell and augment polyfunctional cytotoxic T lymphocyte (CTL) response.

Peptide based vaccine that incorporates one or several highly conserved CD8+ T cells epitopes to induce potent cytotoxic T lymphocyte (CTL) response is desirable for some infectious diseases, such as HIV-1 (human immunodeficiency virus-1), and cancers. However, the CD8+ T cells epitope is often weakly immunogenic, and thus requires a specific adjuvant or delivery system to enhance the efficiency. Here we investigated the use of self-assembling peptide EAK16-II based platform to achieve the co-delivery of CD8+ T cells epitope and TLR7/8 agonists (R848 or R837) for augmenting DCs maturation and HIV-1 specific CTL response. HIV-1 CTL epitope SL9 was conjugated with EAK16-II to obtain SL9-EAK16-II, which further spontaneously co-assembled with R848 or R837 in aqueous solution, forming co-assembled nanofibers. Fluorescence spectra and calorimetrical titration revealed the interaction between SL9-EAK16-II assemblies and R848 or R837 via hydrogen bonding and hydrophobic interaction, with the binding affinity (dissociation constant Kd) of 0.62μM or 0.53μM, respectively. Ex vivo generated DCs from HIV-1+ patients pulsed with the SL9-EAK16-II/R848 nanofibers stimulated significantly more polyfunctional SL9 specific CTLs, compared to the DCs pulsed with SL9 alone or the mixture of SL9 and TLR agonist. Furthermore, the nanofibers elicited stronger SL9 specific CTL response in vaccinated mice. Our findings suggest the self-assembling peptide EAK16-II might be used as a new delivery system for peptide based vaccines.