RESUMO
Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Células Th1/patologia , Células Th17/patologia , Perfilação da Expressão Gênica , Infecções por HIV/virologia , Humanos , Receptores CXCR5/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologia , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Although multiple restriction factors have been shown to inhibit HIV/SIV replication, little is known about their expression in vivo Expression of 45 confirmed and putative HIV/SIV restriction factors was analyzed in CD4+ T cells from peripheral blood and the jejunum in rhesus macaques, revealing distinct expression patterns in naive and memory subsets. In both peripheral blood and the jejunum, memory CD4+ T cells expressed higher levels of multiple restriction factors compared to naive cells. However, relative to their expression in peripheral blood CD4+ T cells, jejunal CCR5+ CD4+ T cells exhibited significantly lower expression of multiple restriction factors, including APOBEC3G, MX2, and TRIM25, which may contribute to the exquisite susceptibility of these cells to SIV infection. In vitro stimulation with anti-CD3/CD28 antibodies or type I interferon resulted in upregulation of distinct subsets of multiple restriction factors. After infection of rhesus macaques with SIVmac239, the expression of most confirmed and putative restriction factors substantially increased in all CD4+ T cell memory subsets at the peak of acute infection. Jejunal CCR5+ CD4+ T cells exhibited the highest levels of SIV RNA, corresponding to the lower restriction factor expression in this subset relative to peripheral blood prior to infection. These results illustrate the dynamic modulation of confirmed and putative restriction factor expression by memory differentiation, stimulation, tissue microenvironment and SIV infection and suggest that differential expression of restriction factors may play a key role in modulating the susceptibility of different populations of CD4+ T cells to lentiviral infection.IMPORTANCE Restriction factors are genes that have evolved to provide intrinsic defense against viruses. HIV and simian immunodeficiency virus (SIV) target CD4+ T cells. The baseline level of expression in vivo and degree to which expression of restriction factors is modulated by conditions such as CD4+ T cell differentiation, stimulation, tissue location, or SIV infection are currently poorly understood. We measured the expression of 45 confirmed and putative restriction factors in primary CD4+ T cells from rhesus macaques under various conditions, finding dynamic changes in each state. Most dramatically, in acute SIV infection, the expression of almost all target genes analyzed increased. These are the first measurements of many of these confirmed and putative restriction factors in primary cells or during the early events after SIV infection and suggest that the level of expression of restriction factors may contribute to the differential susceptibility of CD4+ T cells to SIV infection.
Assuntos
Linfócitos T CD4-Positivos/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Imunidade Inata , Jejuno/imunologia , Jejuno/virologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Transcriptoma/imunologiaRESUMO
UNLABELLED: Despite its importance in shaping adaptive immune responses, viral clearance, and immune-based inflammation, tissue-specific innate immunity remains poorly characterized for hepatitis C virus (HCV) infection due to the lack of access to acutely infected tissues. In this study, we evaluated the impact of natural killer (NK) cells and myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells on control of virus replication and virus-induced pathology caused by another, more rapidly resolving hepacivirus, GB virus B (GBV-B), in infections of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2 to 3 months postinfection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, the levels of NK cells, mDCs, and pDCs in the liver increased up to 3-fold. Although no obvious numerical changes in peripheral innate cells occurred, circulating NK cells exhibited increased perforin and Ki67 expression levels and increased surface expression of CXCR3. These data suggested that increased NK cell arming and proliferation as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed, NK cell frequencies in the liver positively correlated with plasma (R = 0.698; P = 0.015) and liver (R = 0.567; P = 0.057) viral loads. Finally, soluble factors associated with NK cells and DCs, including gamma interferon (IFN-γ) and RANTES, were increased in acute infection and also were associated with viral loads and hepatitis. Collectively, the findings showed that mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis, and potentially to resolution of GBV-B infection, and our results may provide insight into similar mechanisms in HCV infection. IMPORTANCE: Hepatitis C virus (HCV) infection has created a global health crisis, and despite new effective antivirals, it is still a leading cause of liver disease and death worldwide. Recent evidence suggests that innate immunity may be a potential therapeutic target for HCV, but it may also be a correlate of increased disease. Due to a lack of access to human tissues with acute HCV infection, in this study we evaluated the role of innate immunity in resolving infection with a hepacivirus, GBV-B, in common marmosets. Collectively, our data suggest that NK cell and DC mobilization in acute hepacivirus infection can dampen virus replication but also regulate acute and chronic liver damage. How these two opposing effects on the host may be modulated in future therapeutic and vaccine approaches warrants further study.
Assuntos
Células Dendríticas/imunologia , Vírus GB B/imunologia , Hepatite Viral Animal/imunologia , Hepatite Viral Animal/patologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Animais , Callithrix , Citocinas/metabolismo , Vírus GB B/patogenicidade , Fatores Imunológicos/metabolismo , Fígado/patologia , Fígado/virologia , Carga ViralRESUMO
The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVΔnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from naïve animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVΔnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data support the hypothesis that ongoing stimulation by SIVΔnef promotes a distinct protective balance of CD8+ T cell differentiation and activation states.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/imunologia , Fatores de Transcrição/imunologia , Proteínas Virais Reguladoras e Acessórias , Animais , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular/imunologia , Feminino , Perfilação da Expressão Gênica , Macaca mulatta , Vacinas contra a SAIDS/genética , Vírus da Imunodeficiência Símia/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: At least six host-encoded restriction factors (RFs), APOBEC3G, TRIM5α, tetherin, SAMHD1, schlafen 11, and Mx2 have now been shown to inhibit HIV and/or SIV replication in vitro. To determine their role in vivo in the resistance of macaques to mucosally-acquired SIV, we quantified both pre-exposure (basal) and post-exposure mRNA levels of these RFs, Mx1, and IFNγ in PBMC, lymph nodes, and duodenum of rhesus macaques undergoing weekly low dose rectal exposures to the primary isolate, SIV/DeltaB670. RESULTS: Repetitive challenge divided the monkeys into two groups with respect to their susceptibility to infection: highly susceptible (2-3 challenges, 5 monkeys) and poorly susceptible (≥6 challenges, 3 monkeys). Basal RF and Mx1 expression varied among the three tissues examined, with the lowest expression generally detected in duodenal tissues, and the highest observed in PBMC. The one exception was A3G whose basal expression was greatest in lymph nodes. Importantly, significantly higher basal expression of TRIM5α and Mx1 was observed in PBMC of animals more resistant to mucosal infection. Moreover, individual TRIM5α levels were stable throughout a year prior to infection. Post-exposure induction of these genes was also observed after virus appearance in plasma, with elevated levels in PBMC and duodenum transiently occurring 7-10 days post infection. They did not appear to have an effect on control of viremia. Interestingly, minimal to no induction was observed in the resistant animal that became an elite controller. CONCLUSIONS: These results suggest that constitutively expressed TRIM5α appears to play a greater role in restricting mucosal transmission of SIV than that associated with type I interferon induction following virus entry. Surprisingly, this association was not observed with the other RFs. The higher basal expression of TRIM5α observed in PBMC than in duodenal tissues emphasizes the understated role of the second barrier to systemic infection involving the transport of virus from the mucosal compartment to the blood. Together, these observations provide a strong incentive for a more comprehensive examination of the intrinsic, variable control of constitutive expression of these genes in the sexual transmission of HIV.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Duodeno/imunologia , Infecções por HIV/imunologia , Leucócitos Mononucleares/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Duodeno/virologia , Feminino , Infecções por HIV/genética , Infecções por HIV/virologia , Interferon gama/genética , Interferon gama/imunologia , Leucócitos Mononucleares/virologia , Macaca mulatta/genética , Macaca mulatta/imunologia , Macaca mulatta/virologia , Masculino , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/imunologia , RNA Mensageiro/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Natural killer (NK) cells are classically viewed as effector cells that kill virus-infected and neoplastic cells, but recent studies have identified a rare mucosal NK- cell subpopulation secreting the TH17 cytokine IL-22. Here, we report identification of 2 distinct lineages of mucosal NK cells characterized as NKG2A(+)NFIL3(+)RORC(-) and NKp44(+)NFIL3(+)RORC(+). NKG2A(+) NK cells were systemically distributed, cytotoxic, and secreted IFN-γ, whereas NKp44(+) NK cells were mucosae-restricted, noncytotoxic, and produced IL-22 and IL-17. During SIV infection, NKp44(+) NK cells became apoptotic, were depleted, and had an altered functional profile characterized by decreased IL-17 secretion; increased IFN-γ secretion; and, surprisingly, increased potential for cytotoxicity. NKp44(+) NK cells showed no evidence of direct SIV infection; rather, depletion and altered function were associated with SIV-induced up-regulation of inflammatory mediators in the gut, including indoleamine 2,3-dioxygenase 1. Furthermore, treatment of NKp44(+) NK cells with indoleamine 2,3-dioxygenase 1 catabolites in vitro ablated IL-17 production in a dose-dependent manner, whereas other NK-cell functions were unaffected. Thus lentiviral infection both depletes and modifies the functional repertoire of mucosal NK cells involved in the maintenance of gut integrity, a finding that highlights the plasticity of this rare mucosal NK-cell population.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Inflamação/imunologia , Interleucina-17/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linhagem da Célula/imunologia , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Mucosa Gástrica/imunologia , Mucosa Gástrica/metabolismo , Imunidade nas Mucosas/imunologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/virologia , Interferon gama/biossíntese , Interleucina-17/biossíntese , Interleucinas/biossíntese , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Macaca mulatta , Receptor 2 Desencadeador da Citotoxicidade Natural/imunologia , Cultura Primária de Células , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Interleucina 22RESUMO
Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) are essential components in first-line therapy for human immunodeficiency virus (HIV) infection. However, long-term treatment with existing NRTIs can be associated with significant toxic side effects and the emergence of drug-resistant strains. The identification of new NRTIs for the continued management of HIV-infected people therefore is paramount. In this report, we describe the response of a primary isolate of simian immunodeficiency virus (SIV) to 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) both in vitro and in vivo. EFdA was 3 orders of magnitude better than tenofovir (TFV), zidovudine (AZT), and emtricitabine (FTC) in blocking replication of SIV in monkey peripheral blood mononuclear cells (PBMCs) in vitro, and in a preliminary study using two SIV-infected macaques with advanced AIDS, it was highly effective at treating SIV infection and AIDS symptoms in vivo. Both animals had 3- to 4-log decreases in plasma virus burden within 1 week of EFdA therapy (0.4 mg/kg of body weight, delivered subcutaneously twice a day) that eventually became undetectable. Clinical signs of disease (diarrhea, weight loss, and poor activity) also resolved within the first month of treatment. No detectable clinical or pathological signs of drug toxicity were observed within 6 months of continuous therapy. Virus suppression was sustained until drug treatment was discontinued, at which time virus levels rebounded. Although the rebound virus contained the M184V/I mutation in the viral reverse transcriptase, EFdA was fully effective in maintaining suppression of mutant virus throughout the drug treatment period. These results suggest that expanded studies with EFdA are warranted.
Assuntos
Antivirais/farmacologia , Desoxiadenosinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Antivirais/uso terapêutico , Células Cultivadas , Desoxiadenosinas/uso terapêutico , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Emtricitabina , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Macaca mulatta , Masculino , Mutação , Organofosfonatos/farmacologia , DNA Polimerase Dirigida por RNA/metabolismo , Recidiva , Inibidores da Transcriptase Reversa/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Tenofovir , Carga Viral/efeitos dos fármacos , Zidovudina/farmacologiaRESUMO
Human immunodeficiency virus encephalitis causes dementia in acquired immune deficiency syndrome patients. Using proteomic analysis of postmortem cerebrospinal fluid (CSF) and brain tissue from the simian immunodeficiency virus primate model, we demonstrate here a specific increase in YKL-40 that was tightly associated with lentiviral encephalitis. Longitudinal analysis of CSF from simian immunodeficiency virus-infected pigtailed macaques showed an increase in YKL-40 concentration 2 to 8 weeks before death from encephalitis. This increase in YKL-40 correlated with an increase in CSF viral load; it may therefore represent a biomarker for the development of encephalitis. Analysis of banked human CSF from human immunodeficiency virus-infected patients also demonstrated a correlation between YKL-40 concentration and CSF viral load. In vitro studies demonstrated increased YKL-40 expression and secretion by macrophages and microglia but not by neurons or astrocytes. We found that YKL40 displaced extracellular matrix-bound basic fibroblast growth factor (bFGF) as well as inhibited the mitogenic activity of both fibroblast growth factor receptor 1-expressing BaF3 cells and bFGF-induced axonal branching in hippocampal cultures. Taken together, these findings demonstrate that during lentiviral encephalitis, YKL-40 may interfere with the biological activity of bFGF and potentially of other heparin-binding growth factors and chemokines that can affect neuronal function or survival.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Encefalite Viral/líquido cefalorraquidiano , Fator 2 de Crescimento de Fibroblastos/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/líquido cefalorraquidiano , Adipocinas , Animais , Western Blotting , Proteína 1 Semelhante à Quitinase-3 , Encefalite Viral/metabolismo , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular , Glicoproteínas , Infecções por HIV/complicações , Infecções por HIV/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Lectinas , Macaca nemestrina , Espectrometria de Massas , Microglia/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Human NK (hNK) cells play a key role in mediating host immune responses against various infectious diseases. For practical reasons, the majority of the data on hNK cells has been generated using peripheral blood lymphocytes. In contrast, our knowledge of NK cells in human tissues is limited, and not much is known about developmental pathways of hNK cell subpopulations in vivo. Although research in mice has elucidated a number of fundamental features of NK cell biology, mouse, and hNK cells significantly differ in their subpopulations, functions, and receptor repertoires. Thus, there is a need for a model that is more closely related to humans and yet allows experimental manipulations. Non-human primate models offer numerous opportunities for the study of NK cells, including the study of the role of NK cells after solid organ and stem cell transplantation, as well as in acute viral infection. Macaque NK cells can be depleted in vivo or adoptively transferred in an autologous system. All of these studies are either difficult or unethical to carry out in humans. Here we highlight recent advances in rhesus NK cell research and their parallels in humans. Using high-throughput transcriptional profiling, we demonstrate that the human CD56(bright) and CD56(dim) NK cell subsets have phenotypically and functionally analogous counterparts in rhesus macaques. Thus, the use of non-human primate models offers the potential to substantially advance hNK cell research.
RESUMO
Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans.
Assuntos
Vacinas contra a AIDS/farmacologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Vacinas contra a SAIDS/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas de DNA/microbiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Animais , Imunização/métodos , Interferon gama/imunologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/imunologiaRESUMO
Determining the impact of antiretroviral therapy on virus evolution could advance the development of improved therapeutics/vaccines against HIV. Toward this goal, we analyzed virus burden, quasispecies complexity, and T cell responses in SIV/DeltaB670-infected rhesus macaques+/-treatment for 7 months with PMPA (2-30 weeks postinfection). Treatment divided the animals into two groups: poor responders (a reduction of < or =1 log) and responders (> or =2 log reduction) in virus burden. Virus evolution in poor responders and untreated controls was characterized by expression of a complex quasispecies that evolved as the disease progressed. This included the universal loss of a viral genotype selected against by in vitro passage in monkey cells and selected for by propagation in human cells. In contrast, a good response to PMPA was characterized by infection with a less complex quasispecies that evolved more slowly. Interestingly, in 2 of the best responders, the human-preferred genotype persisted until the study was discontinued (89 weeks p.i.). Neither virus burden nor the magnitude of the T cell response at 2 weeks postinfection predicted PMPA responsiveness. However, responders expressed a less complex quasispecies than nonresponders prior to treatment. These data suggest a role for intrinsic host factors in treatment responsiveness, and lend support for therapeutic vaccination as an adjunct to effective therapy.
Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , Organofosfonatos/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/genética , Adenina/farmacologia , Adenina/uso terapêutico , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Farmacorresistência Viral , Evolução Molecular , Genoma Viral , Genótipo , Glicosilação , Análise Heteroduplex , Humanos , Macaca mulatta , Dados de Sequência Molecular , Organofosfonatos/farmacologia , Homologia de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/classificação , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/imunologia , Tenofovir , Carga Viral , Replicação ViralRESUMO
DNA immunization in conjunction with antiretroviral therapy was evaluated in SIV-infected rhesus macaques treated with [R]-9-[2-phosphonylmethoxypropyl]adenine (PMPA). Macaques were immunized monthly with DNA vaccines expressing either SIV gag/tat or SIV gag/tat and 19 CD8+ T cell epitopes during 7 months of therapy. Half the animals from each group were additionally immunized before infection. Only 60% of the animals (4 controls, 20 vaccinated) responded to PMPA (ART responders). All 4 ART responder controls demonstrated viral rebound or CD4 decline after PMPA was withdrawn. In contrast, 17 of 20 vaccinated ART responders contained viral rebound for over 7 months after PMPA was withdrawn. Viral control correlated with stable CD4 counts, higher lymphoproliferation and an increase in the magnitude and breadth of the CD8+ T cell response. Immunizing before infection or with multi-epitopes enhanced these effects. These results demonstrate that DNA immunization during antiretroviral therapy may be an effective strategy to treat HIV infection.
Assuntos
Adenina/análogos & derivados , Organofosfonatos/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas de DNA/administração & dosagem , Viremia/prevenção & controle , Adenina/administração & dosagem , Adenina/uso terapêutico , Animais , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/uso terapêutico , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Progressão da Doença , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene tat/genética , Produtos do Gene tat/imunologia , Imunoterapia Ativa/métodos , Macaca mulatta , Organofosfonatos/uso terapêutico , RNA Viral/sangue , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Estatística como Assunto , Tenofovir , Vacinas de DNA/imunologia , Suspensão de TratamentoRESUMO
Adenovirus serotype 35 (Ad35) is a promising vaccine platform for human immunodeficiency virus (HIV) infection and emerging infectious diseases as it is uncommon in humans worldwide and is distinct from Ad5, the major vaccine serotype for which many individuals have pre-existing immunity. The immunogenicity of a first-generation, replication-competent Ad35-based vaccine was tested in the simian immunodeficiency virus (SIV) rhesus macaque model by evaluating its capacity to boost immunity generated by Ad5-based vectors. A series of four immunizations with replication-defective Ad5 vectors expressing SIVmac239 gag induced high-frequency responses mediated by both CD8(+) and CD4(+) T cells directed against several epitopes. Ad5-specific neutralizing antibody responses that did not neutralize Ad35 were rapidly induced but waned over time. Subsequent immunization with Ad5-based vectors was minimally effective, whereas immunization with Ad35-based vectors generated a strong increase in the frequency of Gag-specific T cells with specificities that were unchanged. While this boosting response was relatively transient, challenge with the distinct pathogenic isolate SIV/DeltaB670 generated robust and selective recall responses to Gag with similar specificities as induced by vaccination that were elevated for 25 weeks relative to controls. Vaccination had measurable albeit minor effects on virus load. Unexpectedly, regional hypervariability within the Gag sequence of SIV/DeltaB670 was associated with mutation of the conserved CD8(+) T-cell epitope CM9 without concurrent flanking mutations and in the absence of immune pressure. These findings support the further development of Ad35 as a vaccine vector, and promote vaccine regimens that utilize serial administration of heterologous adenoviruses.
Assuntos
Adenovírus Humanos/imunologia , Produtos do Gene gag/genética , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/imunologia , Adenovírus Humanos/classificação , Animais , Formação de Anticorpos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Produtos do Gene gag/administração & dosagem , Produtos do Gene gag/sangue , Produtos do Gene gag/imunologia , Vetores Genéticos/imunologia , Imunidade Celular , Imunização Secundária , Macaca mulatta , Vírus da Imunodeficiência Símia/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagemRESUMO
The extent to which simian immunodeficiency virus (SIV) replication in lung tissues contributes to the pool of viruses replicating during acute infection is incompletely understood. To address this issue, in situ hybridization was used to examine SIV replication in multiple lobes of lung from rhesus macaques infected with pathogenic SIV. Despite widespread viral replication in lymphoid and intestinal tissues, the lungs during acute infection harbored rare productively infected cells. Simultaneous immunohistochemical staining for the monocytic marker, CD68, revealed that SIV RNA(+) cells in lung tissues during acute infection were CD68(-), whereas during AIDS they were predominantly CD68(+) and localized in large foci in caudal lobes. SIV RNA(+) cells in spleen remained CD68(-) throughout disease. Since CD68 is also expressed by subpopulations of dendritic cells (DC), we also examined pulmonary CD68(+) cells for expression of additional DC markers. DC-LAMP mRNA was abundant in lung tissues and expressed predominantly by CD68(-) cells, whereas DC-SIGN mRNA was expressed in only very rare cells, indicating that SIV RNA(+) cells late in disease were most likely macrophages. These studies of SIV/host interactions demonstrate that macaque lung tissues are minimally infected during acute infection, exhibit changes in predominant target cells for infection, and express very little DC-SIGN.
Assuntos
Pulmão/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Hibridização In Situ , Macaca mulatta , Especificidade de Órgãos , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Replicação ViralRESUMO
To establish experimental Pneumocystis carinii infection in simian immunodeficiency virus (SIV)-infected macaques as a model of acquired immunodeficiency syndrome (AIDS)-associated P. carinii pneumonia (PCP), SIV-infected macaques were inoculated intrabronchially with macaque-derived P. carinii, and P. carinii-specific polymerase chain reaction (PCR) and flow cytometric analysis of bronchoalveolar lavage fluid were done biweekly for up to 44 weeks after inoculation. All inoculated animals had a P. carinii-specific PCR product after infection. CD8(+) T cells in lung lavage samples from SIV- and P. carinii-coinfected animals increased to >90% of total CD3(+) cells, a pattern associated with naturally acquired P. carinii infection. Progression of disease also was correlated with increased neutrophil infiltration to the lungs. The animals had a protracted period of asymptomatic colonization with P. carinii before progression to PCP. The development of a model of PCP in SIV-infected rhesus macaques provides the means to study AIDS-associated PCP.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/complicações , Pneumocystis , Pneumonia por Pneumocystis/complicações , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Vírus da Imunodeficiência Símia , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/etiologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/virologia , Complexo CD3/análise , Linfócitos T CD8-Positivos/imunologia , DNA Fúngico/análise , Modelos Animais de Doenças , Feminino , Pulmão/microbiologia , Pulmão/patologia , Macaca mulatta , Masculino , Infiltração de Neutrófilos , Pneumocystis/genética , Pneumocystis/isolamento & purificação , Pneumonia por Pneumocystis/diagnóstico , Pneumonia por Pneumocystis/etiologia , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
Chemokines are important mediators of cell trafficking during immune inductive and effector activities, and dysregulation of their expression might contribute to the pathogenesis of human immunodeficiency virus type 1 and the related simian immunodeficiency virus (SIV). To understand better the effects of SIV infection on lymphoid tissues in rhesus macaques, we examined chemokine messenger RNA (mRNA) expression patterns by using DNA filter array hybridization. Of the 34 chemokines examined, the interferon gamma (IFN-gamma)-inducible chemokine CXC chemokine ligand 9/monokine induced by interferon-gamma (CXCL9/Mig) was one of the most highly up-regulated chemokines in rhesus macaque spleen tissue early after infection with pathogenic SIV. The relative levels of expression of CXCL9/Mig mRNA in spleen and lymph nodes were significantly increased after infection with SIV in both quantitative image capture and analysis and real-time reverse transcriptase-polymerase chain reaction assays. In addition, in situ hybridization for CXCL9/Mig mRNA revealed that the patterns of expression were altered after SIV infection. Associated with the increased expression of CXCL9/Mig were increased numbers of IFN-gamma mRNA-positive cells in tissues and reduced percentages of CXC chemokine receptor (CXCR) 3(+)/CD3(+) and CXCR3(+)/CD8(+) lymphocytes in peripheral blood. We propose that SIV replication in vivo initiates IFN-gamma-driven positive-feedback loops in lymphoid tissues that disrupt the trafficking of effector T lymphocytes and lead to chronic local inflammation, thereby contributing to immunopathogenesis.
Assuntos
Quimiocinas CXC/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Linfonodos/metabolismo , Macaca mulatta/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia , Animais , Quimiocina CXCL9 , Quimiocinas CXC/genética , Quimiocinas CXC/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Retroalimentação Fisiológica/imunologia , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/farmacologia , Interferon gama/farmacologia , Linfonodos/patologia , Linfonodos/virologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Distribuição Tecidual , Regulação para Cima/imunologiaRESUMO
An effective vaccine against human immunodeficiency virus (HIV) should protect against mucosal transmission of genetically divergent isolates. As a safe alternative to live attenuated vaccines, the immunogenicity and protective efficacy of a DNA vaccine containing simian immunodeficiency virus (SIV) strain 17E-Fr (SIV/17E-Fr) gag-pol-env was analyzed in rhesus macaques. Significant levels of cytotoxic T lymphocytes (CTL), but low to undetectable serum antibody responses, were observed following multiple immunizations. SIV-specific mucosal antibodies and CTL were also detected in rectal washes and gut-associated lymphoid tissues, respectively. Vaccinated and naive control monkeys were challenged intrarectally with SIV strain DeltaB670 (SIV/DeltaB670), a primary isolate whose env is 15% dissimilar to that of the vaccine strain. Four of seven vaccinees were protected from infection as determined by the inability to identify viral RNA or DNA sequences in the peripheral blood and the absence of anamnestic antibody responses postchallenge. This is the first report of mucosal protection against a primary pathogenic, heterologous isolate of SIV by using a commercially viable vaccine approach. These results support further development of a DNA vaccine for protection against HIV.