RESUMO
Human immunodeficiency virus (HIV)-positive individuals can be superinfected with different virus strains. Individuals who control an initial HIV infection are therefore still at risk for subsequent infection with divergent viruses, but the barriers to such superinfection remain unclear. Here we tested long-term nonprogressors' (LTNPs') susceptibility to superinfection using Indian rhesus macaques that express the major histocompatibility complex class I (MHC-I) allele Mamu-B 17, which is associated with control of the pathogenic AIDS virus SIVmac239. The Mamu-B 17-restricted CD8(+) T cell repertoire is focused almost entirely on 5 epitopes. We engineered a series of SIVmac239 variants bearing mutations in 3, 4, or all 5 of these epitopes and used them to serially challenge 2 Mamu-B 17-positive LTNPs. None of the escape variants caused breakthrough replication in LTNPs, although they readily infected Mamu-B 17-negative naive macaques. In vitro competing coculture assays and examination of viral evolution in hosts lacking Mamu-B 17 suggested that the mutant viruses had negligible defects in replicative fitness. Both LTNPs maintained robust immune responses, including simian immunodeficiency virus (SIV)-specific CD8(+) and CD4(+) T cells and neutralizing antibodies. Our results suggest that escape mutations in epitopes bound by "protective" MHC-I molecules may not be sufficient to establish superinfection in LTNPs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/genética , Sobreviventes de Longo Prazo ao HIV , Macaca mulatta/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Superinfecção/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/virologia , Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Macaca mulatta/virologia , Dados de Sequência Molecular , Mutação , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/classificação , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Superinfecção/virologiaRESUMO
Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT(1A)R)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT(1A)R but are deficient in endogenous angiotensin II receptors (AT(1A)R-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin's endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT(1A)R-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT(1A)R-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT(1A)R-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT(1A)R- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs.
Assuntos
Regulação da Expressão Gênica , Insulina/farmacologia , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Receptor Tipo 1 de Angiotensina/fisiologia , Angiotensina II/farmacologia , Animais , Células Cultivadas , Endocitose/efeitos dos fármacos , Endocitose/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/fisiologia , Túbulos Renais Proximais/efeitos dos fármacos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Receptor Tipo 1 de Angiotensina/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
An effective AIDS vaccine will need to protect against globally diverse isolates of HIV. To address this issue in macaques, we administered a live-attenuated simian immunodeficiency virus (SIV) vaccine and challenged with a highly pathogenic heterologous isolate. Vaccinees reduced viral replication by approximately 2 logs between weeks 2-32 (P < or = 0.049) postchallenge. Remarkably, vaccinees expressing MHC-I (MHC class I) alleles previously associated with viral control completely suppressed acute phase replication of the challenge virus, implicating CD8(+) T cells in this control. Furthermore, transient depletion of peripheral CD8(+) lymphocytes in four vaccinees during the chronic phase resulted in an increase in virus replication. In two of these animals, the recrudescent virus population contained only the vaccine strain and not the challenge virus. Alarmingly, however, we found evidence of recombinant viruses emerging in some of the vaccinated animals. This finding argues strongly against an attenuated virus vaccine as a solution to the AIDS epidemic. On a more positive note, our results suggest that MHC-I-restricted CD8(+) T cells contribute to the protection induced by the live-attenuated SIV vaccine and demonstrate that vaccine-induced CD8(+) T cell responses can control replication of heterologous challenge viruses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Macaca , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral/fisiologia , Animais , Sequência de Bases , Primers do DNA/genética , Variação Genética , Dados de Sequência Molecular , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas contra a SAIDS/genética , Análise de Sequência de RNA , Vírus da Imunodeficiência Símia/genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
The kinetics of peptide presentation by major histocompatibility complex class I (MHC-I) molecules may contribute to the efficacy of CD8+ T cells. Whether all CD8+ T-cell epitopes from a protein are presented by the same MHC-I molecule with similar kinetics is unknown. Here we show that CD8+ T-cell epitopes derived from SIVmac239 Gag are presented with markedly different kinetics. We demonstrate that this discrepancy in presentation is not related to immunodominance but instead is due to differential requirements for epitope generation. These results illustrate that significant differences in presentation kinetics can exist among CD8+ T-cell epitopes derived from the same viral protein.
Assuntos
Apresentação de Antígeno/fisiologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Produtos do Gene gag/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Animais , Células Apresentadoras de Antígenos , Epitopos de Linfócito T/química , Epitopos de Linfócito T/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Epitopos Imunodominantes , Cinética , Vírus da Imunodeficiência Símia/imunologiaRESUMO
The association between particular major histocompatibility complex class I (MHC-I) alleles and control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication implies that certain CD8(+) T-lymphocyte (CD8-TL) responses are better able than others to control viral replication in vivo. However, possession of favorable alleles does not guarantee improved prognosis or viral control. In rhesus macaques, the MHC-I allele Mamu-B*17 is correlated with reduced viremia and is overrepresented in macaques that control SIVmac239, termed elite controllers (ECs). However, there is so far no mechanistic explanation for this phenomenon. Here we show that the chronic-phase Mamu-B*17-restricted repertoire is focused primarily against just five epitopes-VifHW8, EnvFW9, NefIW9, NefMW9, and env(ARF)cRW9-in both ECs and progressors. Interestingly, Mamu-B*17-restricted CD8-TL do not target epitopes in Gag. CD8-TL escape variation occurred in all targeted Mamu-B*17-restricted epitopes. However, recognition of escape variant peptides was commonly observed in both ECs and progressors. Wild-type sequences in the VifHW8 epitope tended to be conserved in ECs, but there was no evidence that this enhances viral control. In fact, no consistent differences were detected between ECs and progressors in any measured parameter. Our data suggest that the narrowly focused Mamu-B*17-restricted repertoire suppresses virus replication and drives viral evolution. It is, however, insufficient in the majority of individuals that express the "protective" Mamu-B*17 molecule. Most importantly, our data indicate that the important differences between Mamu-B*17-positive ECs and progressors are not readily discernible using standard assays to measure immune responses.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/imunologia , Epitopos/química , Epitopos/imunologia , Macaca mulatta/genética , Fases de Leitura/genética , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação Viral/imunologiaRESUMO
Cryptic major histocompatibility complex class I epitopes have been detected in several pathogens, but their importance in the immune response to AIDS viruses remains unknown. Here, we show that Mamu-B*17(+) simian immunodeficiency virus (SIV)mac239-infected rhesus macaques that spontaneously controlled viral replication consistently made strong CD8(+) T lymphocyte (CD8-TL) responses against a cryptic epitope, RHLAFKCLW (cRW9). Importantly, cRW9-specific CD8-TL selected for viral variation in vivo and effectively suppressed SIV replication in vitro, suggesting that they might play a key role in the SIV-specific response. The discovery of an immunodominant CD8-TL response in elite controller macaques against a cryptic epitope suggests that the AIDS virus-specific cellular immune response is likely far more complex than is generally assumed.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Epitopos/imunologia , HIV/imunologia , Vírus da Imunodeficiência Símia/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Animais , Genoma Viral/imunologia , Humanos , Macaca mulatta/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologiaRESUMO
Certain major histocompatibility complex (MHC) class I alleles are associated with the control of human immunodeficiency virus and simian immunodeficiency virus (SIV) replication. We have designed sequence-specific primers for detection of the rhesus macaque MHC class I allele Mamu-B*08 by PCR and screened a cohort of SIV-infected macaques for this allele. Analysis of 196 SIV(mac)239-infected Indian rhesus macaques revealed that Mamu-B*08 was significantly overrepresented in elite controllers; 38% of elite controllers were Mamu-B*08 positive compared to 3% of progressors (P = 0.00001). Mamu-B*08 was also associated with a 7.34-fold decrease in chronic phase viremia (P = 0.002). Mamu-B*08-positive macaques may, therefore, provide a good model to understand the correlates of MHC class I allele-associated immune protection and viral containment in human elite controllers.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral , Alelos , Animais , Sequência de Bases , Testes Genéticos , Antígenos de Histocompatibilidade Classe I/metabolismo , Macaca mulatta , Dados de Sequência Molecular , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Viremia/genética , Viremia/imunologiaRESUMO
"Elite controllers" are individuals that durably control human immunodeficiency virus or simian immunodeficiency virus replication without therapeutic intervention. The study of these rare individuals may facilitate the definition of a successful immune response to immunodeficiency viruses. Here we describe six Indian-origin rhesus macaques that have controlled replication of the pathogenic virus SIVmac239 for 1 to 5 years. To determine which lymphocyte populations were responsible for this control, we transiently depleted the animals' CD8+ cells in vivo. This treatment resulted in 100- to 10,000-fold increases in viremia. When the CD8+ cells returned, control was reestablished and the levels of small subsets of previously subdominant CD8+ T cells expanded up to 2,500-fold above pre-depletion levels. This wave of CD8+ T cells was accompanied by robust Gag-specific CD4 responses. In contrast, CD8+ NK cell frequencies changed no more than threefold. Together, our data suggest that CD8+ T cells targeting a small number of epitopes, along with broad CD4+ T-cell responses, can successfully control the replication of the AIDS virus. It is likely that subdominant CD8+ T-cell populations play a key role in maintaining this control.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Técnicas de Cocultura , Epitopos de Linfócito T/imunologia , Produtos do Gene gag/imunologia , Variação Genética/genética , Células Matadoras Naturais/imunologia , Contagem de Linfócitos , Macaca mulattaRESUMO
Nonhuman primates are widely used to study correlates of protective immunity in AIDS research. Successful cellular immune responses have been difficult to identify because heterogeneity within macaque major histocompatibility complex (MHC) genes results in quantitative and qualitative differences in immune responses. Here we use microsatellite analysis to show that simian immunodeficiency virus (SIV)-susceptible cynomolgus macaques (Macaca fascicularis) from the Indian Ocean island of Mauritius have extremely simple MHC genetics, with six common haplotypes accounting for two-thirds of the MHC haplotypes in feral animals. Remarkably, 39% of Mauritian cynomolgus macaques carry at least one complete copy of the most frequent MHC haplotype, and 8% of these animals are homozygous. In stark contrast, entire MHC haplotypes are rarely conserved in unrelated Indian rhesus macaques. After intrarectal infection with highly pathogenic SIVmac239 virus, a pair of MHC-identical Mauritian cynomolgus macaques mounted concordant cellular immune responses comparable to those previously reported for a pair of monozygotic twins infected with the same strain of human immunodeficiency virus. Our identification of relatively abundant SIV-susceptible, MHC-identical macaques will facilitate research into protective cellular immunity.
Assuntos
Genes MHC da Classe II , Genes MHC Classe I , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia/fisiologia , Animais , Variação Genética , Haplótipos , Macaca , Maurício , Repetições de Microssatélites , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologiaRESUMO
CD8(+) T lymphocytes appear to play a role in controlling human immunodeficiency virus (HIV) replication, yet routine immunological assays do not measure the antiviral efficacy of these cells. Furthermore, it has been suggested that CD8+ T cells that recognize epitopes derived from proteins expressed early in the viral replication cycle can be highly efficient. We used a functional in vitro assay to assess the abilities of different epitope-specific CD8+ T-cell lines to control simian immunodeficiency virus (SIV) replication. We compared the antiviral efficacies of 26 epitope-specific CD8+ T-cell lines directed against seven SIV epitopes in Tat, Nef, Gag, Env, and Vif that were restricted by either Mamu-A*01 or Mamu-A*02. Suppression of SIV replication varied depending on the epitope specificities of the CD8+ T cells and was unrelated to whether the targeted epitope was derived from an early or late viral protein. Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines were consistently superior at suppressing viral replication compared to the other five SIV-specific CD8+ T-cell lines. We also investigated the impact of viral escape on antiviral efficacy by determining if Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines could suppress the replication of an escaped virus. Viral escape abrogated the abilities of Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T cells to control viral replication. However, gamma interferon (IFN-gamma) enzyme-linked immunospot and IFN-gamma/tumor necrosis factor alpha intracellular-cytokine-staining assays detected cross-reactive immune responses against the Gag escape variant. Understanding antiviral efficacy and epitope variability, therefore, will be important in selecting candidate epitopes for an HIV vaccine.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Células Cultivadas , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Produtos do Gene gag/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Macaca mulatta , Mutação Puntual , RNA Viral/sangue , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/química , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Carga Viral , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
The goal of an AIDS vaccine regimen designed to induce cellular immune responses should be to reduce the viral set point and preserve memory CD4 lymphocytes. Here we investigated whether vaccine-induced cellular immunity in the absence of any Env-specific antibodies can control viral replication following multiple low-dose challenges with the highly pathogenic SIVmac239 isolate. Eight Mamu-A*01-positive Indian rhesus macaques were vaccinated with simian immunodeficiency virus (SIV) gag, tat, rev, and nef using a DNA prime-adenovirus boost strategy. Peak viremia (P = 0.007) and the chronic phase set point (P = 0.0192) were significantly decreased in the vaccinated cohort, out to 1 year postinfection. Loss of CD4(+) memory populations was also ameliorated in vaccinated animals. Interestingly, only one of the eight vaccinees developed Env-specific neutralizing antibodies after infection. The control observed was significantly improved over that observed in animals vaccinated with SIV gag only. Vaccine-induced cellular immune responses can, therefore, exert a measure of control over replication of the AIDS virus in the complete absence of neutralizing antibody and give us hope that a vaccine designed to induce cellular immune responses might control viral replication.