RESUMO
BACKGROUND: Varicella-zoster virus (VZV) is under consideration as a promising recombinant viral vector to deliver foreign antigens including HIV. However, new vectors have come under increased scrutiny, since trials with adenovirus serotype 5-vectored (Ad5-vectored) HIV vaccine demonstrated increased HIV risk in individuals with pre-immunity to the vector that was thought to be associated with mucosal immune activation (IA). Therefore, given the prospect of developing an HIV/VZV chimeric vaccine, it is particularly important to define the impact of VZV vaccination on IA. METHODS: Healthy VZV-seropositive Kenyan women (n = 44) were immunized with high-dose live attenuated VZV vaccine, and we assessed the expression on CD4+ T cells isolated from blood, cervix, and rectum of IA markers including CD38 and HLA-DR and of markers of cell migration and tissue retention, as well as the concentration of genital and intestinal cytokines. A delayed-start group (n = 22) was used to control for natural variations in these parameters. RESULTS: Although immunogenic, VZV vaccination did not result in significant difference in the frequency of cervical activated (HLA-DR+CD38+) CD4+ T cells (median 1.61%, IQR 0.93%-2.76%) at 12 weeks after vaccination when compared with baseline (median 1.58%, IQR 0.75%-3.04%), the primary outcome for this study. VZV vaccination also had no measurable effect on any of the IA parameters at 4, 8, and 12 weeks after vaccination. CONCLUSION: This study provides the first evidence to our knowledge about the effects of VZV vaccination on human mucosal IA status and supports further evaluation of VZV as a potential vector for an HIV vaccine. TRIAL REGISTRATION: ClinicalTrials.gov NCT02514018. FUNDING: Primary support from the Canadian Institutes for Health Research (CIHR). For other sources, see Acknowledgments.
Assuntos
Vacinas contra a AIDS , Linfócitos T CD4-Positivos/imunologia , HIV-1/fisiologia , Herpesvirus Humano 3 , Ativação Viral , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Adulto , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Feminino , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/imunologia , Humanos , Quênia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Infecção pelo Vírus da Varicela-Zoster/imunologia , Infecção pelo Vírus da Varicela-Zoster/patologia , Infecção pelo Vírus da Varicela-Zoster/prevenção & controle , Ativação Viral/efeitos dos fármacos , Ativação Viral/imunologiaRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection often arises from a single transmitted/founder (TF) viral variant among a large pool of viruses in the quasispecies in the transmitting partner. TF variants are typically nondominant in blood and genital secretions, indicating that they have unique traits. The plasmacytoid dendritic cell (pDC) is the primary alpha interferon (IFN-α)-producing cell in response to viral infections and is rapidly recruited to the female genital tract upon exposure to HIV-1. The impact of pDCs on transmission is unknown. We investigated whether evasion of pDC responses is a trait of TF viruses. pDCs from healthy donors were stimulated in vitro with a panel of 20 HIV-1 variants, consisting of one TF variant and three nontransmitted (NT) variants each from five transmission-linked donor pairs, and secretion of IFN-α and tumor necrosis factor alpha (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA). No significant differences in cytokine secretion in response to TF and NT viruses were observed, despite a trend toward enhanced IFN-α and TNF-α production in response to TF viruses. NT viruses demonstrated polarization toward production of either IFN-α or TNF-α, indicating possible dysregulation. Also, for NT viruses, IFN-α secretion was associated with increased resistance of the virus to inactivation by IFN-α in vitro, suggesting in vivo evolution. Thus, TF viruses do not appear to preferentially subvert pDC activation compared to that with nontransmitted HIV-1 variants. pDCs may, however, contribute to the in vivo evolution of HIV-1.IMPORTANCE The plasmacytoid dendritic cell (pDC) is the first cell type recruited to the site of HIV-1 exposure; however, its contribution to the viral bottleneck in HIV-1 transmission has not been explored previously. We hypothesized that transmitted/founder viruses are able to avoid the pDC response. In this study, we used previously established donor pair-linked transmitted/founder and nontransmitted (or chronic) variants of HIV-1 to stimulate pDCs. Transmitted/founder HIV-1, instead of suppressing pDC responses, induced IFN-α and TNF-α secretion to levels comparable to those induced by viruses from the transmitting partner. We noted several unique traits of chronic viruses, including polarization between IFN-α and TNF-α production as well as a strong relationship between IFN-α secretion and the resistance of the virus to neutralization. These data rule out the possibility that TF viruses preferentially suppress pDCs in comparison to the pDC response to nontransmitted HIV variants. pDCs may, however, be important drivers of viral evolution in vivo.
Assuntos
Células Dendríticas/imunologia , Infecções por HIV/transmissão , HIV-1/imunologia , Interferon-alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Células Dendríticas/virologia , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Interferon-alfa/imunologia , Masculino , Testes de Neutralização , Cultura Primária de Células , Fator de Necrose Tumoral alfa/imunologia , Vírion/imunologia , Vírion/patogenicidadeRESUMO
The capacity of HIV-1 to develop resistance to current drugs calls for innovative strategies to control this infection. We aimed at developing novel inhibitors of HIV-1 replication by targeting viral RNA processing-a stage dependent on conserved host processes. We previously reported that digoxin is a potent inhibitor of this stage. Herein, we identify 12 other cardiac glycoside/aglycones or cardiotonic steroids (CSs) that impede HIV growth in HIV-infected T cells from clinical patients at IC50s (1.1-1.3 nM) that are 2-26 times below concentrations used in patients with heart conditions. We subsequently demonstrate that CSs inhibit HIV-1 gene expression in part through modulation of MEK1/2-ERK1/2 signaling via interaction with the Na+/K+-ATPase, independent of alterations in intracellular Ca2+. Supporting this hypothesis, depletion of the Na+/K+-ATPase or addition of a MEK1/2-ERK1/2 activator also impairs HIV-1 gene expression. Similar to digoxin, all CSs tested induce oversplicing of HIV-1 RNAs, reducing unspliced (Gag) and singly spliced RNAs (Env/p14-Tat) encoding essential HIV-1 structural/regulatory proteins. Furthermore, all CSs cause nuclear retention of genomic/unspliced RNAs, supporting viral RNA processing as the underlying mechanism for their disruption of HIV-1 replication. These findings call for further in vivo validation and supports the targeting of cellular processes to control HIV-1 infection.
Assuntos
Glicosídeos Cardíacos/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Glicosídeos Cardíacos/química , Digoxina/química , Digoxina/farmacologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Replicação Viral/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a "regulatory" M2 phenotype or an "inflammatory" M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more "regulatory" stimuli such as TGF-ß/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.
Assuntos
Ouro/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Nanopartículas Metálicas/química , Ouro/sangue , Ouro/química , Humanos , Fígado/citologia , Macrófagos/química , Monócitos/química , Monócitos/metabolismo , FenótipoRESUMO
Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.
Assuntos
Antivirais/farmacologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Proteínas/farmacologia , Linfócitos T Citotóxicos/imunologia , Latência Viral/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Humanos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão , Ativação Viral/efeitos dos fármacosRESUMO
T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Tolerância Imunológica/imunologia , Proteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD/química , Antígenos CD/imunologia , Autoimunidade/imunologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Neoplasias Colorretais/imunologia , Modelos Animais de Doenças , Feminino , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Inflamação/imunologia , Inflamação/patologia , Ligantes , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mucosa/imunologia , Mucosa/patologia , Conformação Proteica , Multimerização Proteica , Receptores Virais/química , Receptores Virais/imunologiaRESUMO
The T cell Ig- and mucin domain-containing molecule-3 (Tim-3) negative immune checkpoint receptor demarcates functionally exhausted CD8(+) T cells arising from chronic stimulation in viral infections like HIV. Tim-3 blockade leads to improved antiviral CD8(+) T cell responses in vitro and, therefore, represents a novel intervention strategy to restore T cell function in vivo and protect from disease progression. However, the Tim-3 pathway in the physiologically relevant rhesus macaque SIV model of AIDS remains uncharacterized. We report that Tim-3(+)CD8(+) T cell frequencies are significantly increased in lymph nodes, but not in peripheral blood, in SIV-infected animals. Tim-3(+)PD-1(+)CD8(+) T cells are similarly increased during SIV infection and positively correlate with SIV plasma viremia. Tim-3 expression was found primarily on effector memory CD8(+) T cells in all tissues examined. Tim-3(+)CD8(+) T cells have lower Ki-67 content and minimal cytokine responses to SIV compared with Tim-3(-)CD8(+) T cells. During acute-phase SIV replication, Tim-3 expression peaked on SIV-specific CD8(+) T cells by 2 wk postinfection and then rapidly diminished, irrespective of mutational escape of cognate Ag, suggesting non-TCR-driven mechanisms for Tim-3 expression. Thus, rhesus Tim-3 in SIV infection partially mimics human Tim-3 in HIV infection and may serve as a novel model for targeted studies focused on rejuvenating HIV-specific CD8(+) T cell responses.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunoterapia/métodos , Proteínas de Membrana/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Síndrome da Imunodeficiência Adquirida/terapia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/virologia , Proliferação de Células , Células Cultivadas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Memória Imunológica , Macaca mulatta , Proteínas de Membrana/genética , Dados de Sequência Molecular , Terapia de Alvo Molecular , Receptor de Morte Celular Programada 1/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Carga Viral , Replicação ViralRESUMO
Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis), such as suberanilohydroxamic acid (SAHA), romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL). Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i) the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii) the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Depsipeptídeos/farmacologia , Infecções por HIV/imunologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Linfócitos T Citotóxicos/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Panobinostat , Linfócitos T Citotóxicos/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The enormous sequence diversity of HIV remains a major roadblock to the development of a prophylactic vaccine and new approaches to induce protective immunity are needed. Endogenous retrotransposable elements (ERE) such as endogenous retrovirus K (ERV)-K and long interspersed nuclear element-1 (LINE-1) are activated during HIV-1-infection and could represent stable, surrogate targets to eliminate HIV-1-infected cells. Here, we explored the hypothesis that vaccination against ERE would protect macaques from acquisition and replication of simian immunodeficiency virus (SIV). Following vaccination with antigens derived from LINE-1 and ERV-K consensus sequences, animals mounted immune responses that failed to delay acquisition of SIVsmE660. We observed no differences in acute or set point viral loads between ERE-vaccinated and control animals suggesting that ERE-specific responses were not protective. Indeed, ERE-specific T cells failed to expand anamnestically in vivo following infection with SIVsmE660 and did not recognize SIV-infected targets in vitro, in agreement with no significant induction of targeted ERE mRNA by SIV in macaque CD4+ T cells. Instead, lower infection rates and viral loads correlated significantly to protective TRIM5α alleles. Cumulatively, these data demonstrate that vaccination against the selected ERE consensus sequences in macaques did not lead to immune-mediated recognition and killing of SIV-infected cells, as has been shown for HIV-infected human cells using patient-derived HERV-K-specific T cells. Thus, further research is required to identify the specific nonhuman primate EREs and retroviruses that recapitulate the activity of HIV-1 in human cells. These results also highlight the complexity in translating observations of the interplay between HIV-1 and human EREs to animal models.
Assuntos
Sequência Consenso , Macaca mulatta/imunologia , Retroelementos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Vacinação , Replicação Viral/fisiologia , Animais , Proliferação de Células , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Macaca mulatta/virologia , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Ubiquitina-Proteína LigasesRESUMO
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-3(hi)CD8(+) 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.
Assuntos
Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Sinapses/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Galectinas/metabolismo , Infecções por HIV/metabolismo , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Antígenos Comuns de Leucócito/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4⺠T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific Opa(CEA), but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, Opa(CEA) binding to CEACAM1 reduced the DCs' capacity to stimulate an allogeneic T cell proliferative response. Moreover, Opa(CEA)-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with Opa(CEA)-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.
Assuntos
Antígenos CD/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , HIV-1/imunologia , Neisseria gonorrhoeae/metabolismo , Linfócitos T Citotóxicos/imunologia , Imunidade Adaptativa , Proliferação de Células , Células Dendríticas/microbiologia , Células Dendríticas/virologia , Regulação para Baixo , Fímbrias Bacterianas/fisiologia , Humanos , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Monócitos/citologia , Neisseria gonorrhoeae/fisiologia , Especificidade da Espécie , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/virologia , Antígeno CD83RESUMO
BACKGROUND: An estimated 10-20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial. RESULTS: Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets. CONCLUSIONS: Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology.
Assuntos
Retrovirus Endógenos/imunologia , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Infecções por HTLV-I/imunologia , Linfócitos T/imunologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/virologia , Retrovirus Endógenos/fisiologia , HIV-1/fisiologia , HIV-2/fisiologia , Imunidade Celular , Vírus da Imunodeficiência Símia/fisiologia , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Retrovirus Endógenos/imunologia , Retrovirus Endógenos/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene gag/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/isolamento & purificação , HIV-2/imunologia , HIV-2/isolamento & purificação , Interações Hospedeiro-Patógeno , Humanos , Dados de Sequência Molecular , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/isolamento & purificação , Ativação Transcricional , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Integração Viral , Internalização do Vírus , Produtos do Gene vif do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
Human endogenous retroviruses (HERV) are remnants of ancestral retroviral infections integrated into the germ line, and constitute approximately 8% of the genome. Several autoimmune disorders, malignancies, and infectious diseases such as HIV-1 are associated with higher HERV expression. The degree to which HERV expression in vivo results in persistent inflammation is not known. We studied the association of immune activation and HERV-K expression in 20 subjects with chronic, untreated progressive HIV-1 infection and 10 HIV-1 negative controls. The mean HERV-K gag and env RNA expression level in the HIV-1 infected cohort was higher than in the control group (p = 0.0003), and was negatively correlated with the frequency of activated CD38+HLA-DR+CD4+ T cells (Rho = -0.61; p = 0.01) and activated CD38+HLA-DR+CD8+ T cells (Rho = -0.51; p = 0.03). Although HIV-infected persons had higher levels of HERV-K RNA expression (as expected), the level of RNA expression was negatively associated with level of T cell activation. The mechanism for this unexpected association remains to be defined.
Assuntos
Retrovirus Endógenos/genética , Retrovirus Endógenos/imunologia , Regulação Viral da Expressão Gênica , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Imunidade/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Estudos de Casos e Controles , Demografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , RNA Viral/metabolismoRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/imunologia , Proteínas de Membrana/metabolismo , Anticorpos/imunologia , Anticorpos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Imunofenotipagem , Proteínas de Membrana/imunologia , Perforina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas com Domínio T/metabolismoRESUMO
The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Elementos de DNA Transponíveis/imunologia , Retrovirus Endógenos/imunologia , Vacinas contra a AIDS/genética , Adulto , Sequência de Aminoácidos , Animais , Vacinas Anticâncer/genética , Elementos de DNA Transponíveis/genética , Modelos Animais de Doenças , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
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-1ß 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.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , HIV/imunologia , HIV/fisiologia , Interferon-alfa/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células Dendríticas/metabolismo , Humanos , Orthomyxoviridae/imunologia , Orthomyxoviridae/fisiologia , Fosforilação , Especificidade da Espécie , Quinase Syk , Fatores de Tempo , Replicação ViralRESUMO
Neisseria gonorrhoeae, the cause of the sexually transmitted infection gonorrhea, elicits low levels of specific Ig that decline rapidly after the bacteria are cleared. Reinfection with the same serovar can occur, and prior gonococcal infection does not alter the Ig response upon subsequent exposure, suggesting that protective immunity is not induced. The mucosal Ig response apparent during gonorrhea does not correlate with that observed systemically, leading to a suggestion that it is locally generated. In considering whether N. gonorrhoeae directly influences B cells, we observed that gonococcal infection prolonged viability of primary human B cells in vitro and elicited robust activation and vigorous proliferative responses in the absence of T cells. Furthermore, we observed the specific expansion of IgD(+)CD27(+) B cells in response to gonococcal infection. These cells are innate in function, conferring protection against diverse microbes by producing low-affinity, broadly reactive IgM without inducing classical immunologic memory. Although gonococcal infection of B cells produced small amounts of gonococcal-specific IgM, IgM specific for irrelevant Ags were also produced, suggesting a broad, polyspecific Ig response. The gonococci were effectively bound and engulfed by B cells. TLR9-inhibitory CpGs blocked B cell responses, indicating that intracellular bacterial degradation allows for innate immune detection within the phagolysosome. To our knowledge, this is the first report of a bacterial pathogen having specific affinity for the human IgM memory B cells, driving their potent activation and polyclonal Ig response. This unfocused T-independent response explains the localized Ig response that occurs, despite an absence of immunologic memory elicited during gonorrhea.
Assuntos
Anticorpos Antibacterianos/imunologia , Linfócitos B/imunologia , Imunidade Inata , Imunoglobulina M/biossíntese , Neisseria gonorrhoeae/imunologia , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Linfócitos B/microbiologia , Proliferação de Células , Células Cultivadas , Gonorreia/imunologia , Gonorreia/microbiologia , Humanos , Imunoglobulina D/biossíntese , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Memória Imunológica , Masculino , Oligonucleotídeos/farmacologia , Fagocitose , Fagossomos/imunologia , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15 mer 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 15 mer peptide mapping.
Assuntos
Retrovirus Endógenos/imunologia , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Mapeamento de Peptídeos/métodos , Linfócitos T/imunologia , Retrovirus Endógenos/genética , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Humanos , RNA Viral/genéticaRESUMO
The signaling adaptor TNFR-associated factor 1 (TRAF1) is specifically lost from virus-specific CD8 T cells during the chronic phase of infection with HIV in humans or lymphocytic choriomeningitis virus (LCMV) clone 13 in mice. In contrast, TRAF1 is maintained at higher levels in virus-specific T cells of HIV controllers or after acute LCMV infection. TRAF1 expression negatively correlates with programmed death 1 expression and HIV load and knockdown of TRAF1 in CD8 T cells from viral controllers results in decreased HIV suppression ex vivo. Consistent with the desensitization of the TRAF1-binding co-stimulatory receptor 4-1BB, 4-1BBL-deficient mice have defects in viral control early, but not late, in chronic infection. TGFß induces the posttranslational loss of TRAF1, whereas IL-7 restores TRAF1 levels. A combination treatment with IL-7 and agonist anti-4-1BB antibody at 3 wk after LCMV clone 13 infection expands T cells and reduces viral load in a TRAF1-dependent manner. Moreover, transfer of TRAF1(+) but not TRAF1(-) memory T cells at the chronic stage of infection reduces viral load. These findings identify TRAF1 as a potential biomarker of HIV-specific CD8 T cell fitness during the chronic phase of disease and a target for therapy.