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Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication-defective adenovirus (rAd) vaccine vectors are at the forefront of this work and have been tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV Gag or other nonenvelope proteins induces T cell responses that control virus load but disappointingly is unsuccessful so far in preventing infection, and attention has turned to inducing antibodies to the envelope. However, here we report that Mauritian cynomolgus macaques (MCM), Macaca fascicularis, vaccinated with unmodified SIV gag alone in a DNA prime followed by an rAd boost exhibit increased protection from infection by repeated intrarectal challenge with low-dose SIVmac251. There was no evidence of infection followed by eradication. A significant correlation was observed between cytokine expression by CD4 T cells and delayed infection. Vaccination with gag fused to the ubiquitin gene or fragmented, designed to increase CD8 magnitude and breadth, did not confer resistance to challenge or enhance immunity. On infection, a significant reduction in peak virus load was observed in all vaccinated animals, including those vaccinated with modified gag These findings suggest that a nonpersistent viral vector vaccine coding for internal virus proteins may be able to protect against HIV type 1 (HIV-1) infection. The mechanisms are probably distinct from those of antibody-mediated virus neutralization or cytotoxic CD8 cell killing of virus-infected cells and may be mediated in part by CD4 T cells.IMPORTANCE The simian immunodeficiency virus (SIV) macaque model represents the best animal model for testing new human immunodeficiency virus type 1 (HIV-1) vaccines. Previous studies employing replication-defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with an rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to the envelope and neutralize the virus or mediate antibody-dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established has also shown some success. In contrast, the vaccine described here mediates resistance by a different mechanism from that described above, which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development.
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Produtos do Gene gag/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vírus Defeituosos/genética , Vírus Defeituosos/imunologia , Produtos do Gene gag/genética , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Macaca fascicularis , Masculino , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Carga ViralRESUMO
Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.
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Adenoviridae/imunologia , Antígenos CD/fisiologia , Linfócitos T CD8-Positivos/imunologia , Lectinas Tipo C/fisiologia , Lectinas de Ligação a Manose/fisiologia , Agulhas , Pele , Vacinas Virais/imunologia , Adenoviridae/genética , Citometria de Fluxo , Vetores Genéticos , Microscopia ConfocalRESUMO
Tebentafusp is a gp100xCD3 bispecific ImmTAC designed to redirect polyclonal T cells against cells presenting the melanocyte lineage specific antigen gp100 on HLA-A*02:01. Skin-related adverse events, predominantly rash, are frequent and occur within a few hours after initial infusions, yet the mechanisms are unknown. Here we analysed clinical data from the randomised phase 3 trial (NCT03070392) of tebentafusp (n=252) versus investigator's choice (n=126). Translational analyses were performed on paired on-treatment skin samples from 19 patients collected in the phase 1 trial (NCT01211262). Our analyses showed that rash is a clinical manifestation of tebentafusp-induced recruitment of T cells to cutaneous melanocytes. Development of rash depended on baseline expression levels of gp100 and other melanin pathway genes in the skin. On treatment, melanocyte number was reduced and expression of melanocytic genes decreased, while gene expression related to immunity and cytokine signalling increased. When adjusted for baseline prognostic features, patients with rash within the first week of tebentafusp treatment had the same overall survival compared to patients without a rash in the phase 3 randomized trial IMCgp100-202 (HR 0.84; 95% CI 0.53-1.32). In summary, skin rash is an off-tumour, on-target effect of tebentafusp against gp100+ melanocytes, in line with the mechanism of action.
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In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing alpha(4)beta(7) integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-gamma production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.
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Adenoviridae/imunologia , Linfócitos T CD4-Positivos/imunologia , HIV-1/imunologia , Imunidade nas Mucosas/imunologia , Memória Imunológica/imunologia , Vacinação , Vacinas contra a AIDS/imunologia , Adenoviridae/genética , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/citologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Infecções por HIV/imunologia , HIV-1/patogenicidade , Humanos , Integrina alfa4/imunologia , Cadeias beta de Integrinas/imunologia , Ativação Linfocitária/imunologia , Mucosa/imunologia , Fenótipo , Receptores CCR/imunologia , Receptores CCR4/imunologiaRESUMO
The best hope of controlling the HIV pandemic is the development of an effective vaccine. In addition to the stimulation of virus neutralising antibodies, a vaccine will need an effective T-cell response against the virus. Vaccines based on recombinant adenoviruses (rAd) are promising candidates to stimulate anti-HIV T-cell responses. This review discusses the different rAd vector types, problems raised by host immune responses against them and strategies that are being adopted to overcome this problem. Vaccines need to target and stimulate dendritic cells and thus the tropism and interaction of rAd-based vaccines with these cells is covered. Different rAd vaccination regimes and the need to stimulate mucosal responses are discussed together with data from animal studies on immunogenicity and virus challenge experiments. The review ends with a discussion of the recent disappointing Merck HIV vaccine trial.
Assuntos
Vacinas contra a AIDS/imunologia , Adenoviridae/imunologia , Vetores Genéticos/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas contra a AIDS/genética , Adenoviridae/genética , Animais , Ensaios Clínicos como Assunto , Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Imunidade nas Mucosas , Linfócitos T/imunologia , Linfócitos T/virologia , Resultado do Tratamento , Vacinação/métodos , Vacinas Sintéticas/imunologiaRESUMO
Human plasmacytoid dendritic cells secrete high levels of IFNα and are thus implicated in the activation of NK cells. Activated NK cells are characterised by the up-regulation of CD69 and MHC class II DR expression, secretion of IFN γ and enhanced cytotoxicity. We show that pDC mediate these processes by different mechanisms, some of which overlap. Human NK cells were analysed after co-culture with immature or CpG-matured blood pDC or with supernatant from these cells. Maximal CD69 expression by NK cells was mediated by supernatant from mature pDC and did not require pDC contact. Up-regulation was due in part to IFNα but also to factors in IFNα negative supernatant from immature DC. HLA-DR expression was independent of secreted molecules but required contact with immature or mature DC. Enhanced NK cytotoxicity, measured by killing of K562 targets and expression of CD107a, was mediated by multiple factors including type I IFN, supernatant from immature pDC cultures and contact with immature or mature pDC. These factors act cumulatively to enhance cytotoxcity. Thus different parameters of pDC mediated NK cell activation are regulated by distinct pathways.
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Langerhans' cells (LC) and dermal dendritic cells (dDC) are located in the superficial and deeper layers of the skin respectively and represent the main dendritic cell (DC) populations of the skin. LC-like and dDC-like DC can be generated from CD34 stem cells and this system is widely used as a model for investigating these cells and in therapeutic vaccination. Here we report toll-like receptor (TLR) expression in human LC and dDC derived from CD34 stem cells. In vitro-generated DC expressed TLR-1, 2, 4, 6, 8 and 10. LC, but not dDC, expressed TLR-5, whereas only dDC expressed TLR-3. Maturation of LC was mediated by TLR-2, 4 and 5 ligands, but not by a TLR-3 ligand. dDC maturation was induced by TLR-3 and -4, but not with TLR-5 ligand and only weakly by a TLR-2 ligand. Stimulated LC secreted interleukin (IL)-1beta, low levels of tumour necrosis factor-alpha (TNF-alpha) and IL-8, but not IL-6 or IL-10. dDC secreted TNF-alpha, IL-6, IL-8 and IL-10, but little IL-1beta. IL-12p70 was not produced by ligand-stimulated dDC or LC, but was secreted by monocyte-derived DC (mdDC) stimulated with lipopolysaccharide (LPS). Thus, in vitro-generated LC and dDC detect different pathogen-associated molecules and show different cytokine-secretion profiles in response to TLR ligands.
Assuntos
Citocinas/metabolismo , Células de Langerhans/imunologia , Células-Tronco/imunologia , Receptores Toll-Like/metabolismo , Antígenos CD34/análise , Diferenciação Celular/imunologia , Células Cultivadas , Antígenos HLA-DR/biossíntese , Humanos , Imunofenotipagem , Células de Langerhans/citologia , Ligantes , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células-Tronco/citologia , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima/imunologiaRESUMO
During pregnancy, the mother allows the immunologically distinct fetoplacental unit to develop and grow. Opinions are divided as to whether this represents a state of fetal-specific tolerance or of a generalized suppression of the maternal immune system. We hypothesized that antigen-specific T cell responses are modulated by an inhibitory T cell phenotype and modified dendritic cell (DC) phenotype in a gestation-dependent manner. We analyzed changes in surface markers of peripheral blood T cells, ex vivo antigen-specific T cell responses, indoleamine 2,3-dioxygenase (IDO) activity (kynurenine/tryptophan ratio, KTR), plasma neopterin concentration, and the in vitro expression of progesterone-induced blocking factor (PIBF) in response to peripheral blood mononuclear cell culture with progesterone. We found that mid gestation is characterized by reduced antigen-specific T cell responses associated with (1) predominance of effector memory over other T cell subsets; (2) upregulation of inhibitory markers (programmed death ligand 1); (3) heightened response to progesterone (PIBF); and (4) reduced proportions of myeloid DC and concurrent IDO activity (KTR). Conversely, antigen-specific T cell responses normalized in late pregnancy and were associated with increased markers of T cell activation (CD38, neopterin). However, these changes occur with a simultaneous upregulation of immune suppressive mechanisms including apoptosis (CD95), coinhibition (TIM-3), and immune regulation (IL-10) through the course of pregnancy. Together, our data suggest that immune tolerance dominates in the second trimester and that it is gradually reversed in the third trimester in association with immune activation as the end of pregnancy approaches.
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A major obstacle to therapy in intensive care units is sepsis caused by severe infection. In recent years gram-positive (G+) bacteria, most commonly staphylococci, are thought to be the main pathogens. Micheliolide (MCL) was demonstrated to provide a therapeutic role in rheumatoid arthritis, inflammatory intestinal disease, colitis-associated cancer, and lipopolysaccharide (LPS, the main component of G- bacterial cell wall) induced septic shock. We proved here that MCL played an anti-inflammatory role in Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) induced peritonitis. It inhibited the expression of inflammatory cytokines and chemokines in macrophages and dendritic cells upon stimulation with peptidoglycan (PGN, the main cell wall composition of G+ bacteria). PI3K/Akt and NF-κB pathways account for the anti-inflammatory role of MCL after PGN stimulation. MCL reduced IL-6 secretion through down-regulating NF-κB activation and improved the survival status in mice challenged with a lethal dose of S. aureus. In MRSA infection mouse model, MCL down-regulated the expression of IL-6, TNF-α, MCP-1/CCL2 and IFN-γ in sera, and ameliorated the organ damage of liver and kidney. In conclusion, MCL can help maintain immune equilibrium and decrease PGN, S. aureus and MRSA-triggered inflammatory response. These provide the rationality for the potential usage of MCL in sepsis caused by G+ bacteria (e.g., S. aureus) and antibiotic-resistant bacteria (e.g., MRSA).
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Sesquiterpenos de Guaiano/farmacologia , Transdução de Sinais/efeitos dos fármacos , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/efeitos dos fármacos , Animais , Citocinas/metabolismo , Feminino , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Sepse/imunologia , Sepse/microbiologia , Sepse/prevenção & controle , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologiaRESUMO
In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues.
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Influenza Humana/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Virus da Influenza A Subtipo H5N1 , Influenza Humana/patologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fenótipo , Carga Viral , Adulto JovemRESUMO
SAMHD1 is a restriction factor for HIV-1 infection. SAMHD1 mutations cause the autoinflammatory Aicardi-Goutières syndrome that is characterized by chronic type I interferon (IFN) secretion. We show that the spontaneous IFN response in SAMHD1-deficient cells and mice requires the cGAS/STING cytosolic DNA-sensing pathway. We provide genetic evidence that cell-autonomous control of lentivirus infection in myeloid cells by SAMHD1 limits virus-induced production of IFNs and the induction of co-stimulatory markers. This program of myeloid cell activation required reverse transcription, cGAS and STING, and signaling through the IFN receptor. Furthermore, SAMHD1 reduced the induction of virus-specific cytotoxic T cells in vivo. Therefore, virus restriction by SAMHD1 limits the magnitude of IFN and T cell responses. This demonstrates a competition between cell-autonomous virus control and subsequent innate and adaptive immune responses, a concept with important implications for the treatment of infection.
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Imunidade Adaptativa/imunologia , HIV-1/imunologia , Imunidade Inata/imunologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Animais , Células Dendríticas/imunologia , Infecções por HIV/metabolismo , HIV-1/genética , Interferon beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 1 com Domínio SAM e Domínio HD/deficiência , Replicação Viral/imunologiaRESUMO
Adaptive immune responses are initiated by resident myeloid tissue DC. A major fraction of tissue DC express CD1c+ and is thought to be derived from blood CD1c DC, an idea supported here by the observation that they express tissue-homing molecules and rapidly differentiate into cells with a tissue DC phenotype. Responses are thought to be augmented/modulated further by inflammatory moDC. Although much accepted human myeloid DC cell biology is based on moDC studies, we find these 2 DC populations to be functionally distinct. Stimulated moDC produce high levels of IL-10 and the Th1-promoting cytokine IL-12. Under identical conditions, CD1c DC synthesized no IL-10 and no or low levels of IL-12. Despite this, CD1c DC stimulated a strong Th1 response, demonstrated by IL-12 neutralization to be IL-12 independent, whereas the response induced by moDC was IL-12 dependent. This finding was supported by studies on a patient with a highly reduced ability to synthesize IL-12, whose CD1c DC induced a good Th1 response contrasting with the failure of his moDC, which were impaired in IL-12 production, to induce IFN-γ-secreting T cells. The IL-10 and IL-12 data were confirmed by microarray analysis, which also showed that stimulated moDC produced inflammatory-associated chemokines and cytokines, whereas stimulated CD1c DC showed minimal up-regulation of these genes. Thus, moDC, widely used as a human myeloid DC model, do not faithfully reflect the properties of CD1c tissue DC, making the initial response to a pathogen or vaccine.
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Although the innate immune system is implicated in the control of Kaposi's sarcoma (KS), the risk of developing KS is not associated with the nadir natural killer (NK) cell count, and NK cell counts do not significantly increase or decrease during KS resolution. KS-associated herpesvirus replication was not demonstrated in vivo or in vitro within NK cells, suggesting that NK cells do not contribute to the resolution of KS. Their role appears limited to events occurring during early infection.
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Infecções por HIV/complicações , Herpesvirus Humano 8/isolamento & purificação , Células Matadoras Naturais/virologia , Sarcoma de Kaposi/imunologia , Terapia Antirretroviral de Alta Atividade , Seguimentos , Infecções por HIV/tratamento farmacológico , Humanos , Células Matadoras Naturais/imunologia , Contagem de Leucócitos , Medição de Risco , Sarcoma de Kaposi/virologiaRESUMO
The efficient induction of CD8 T cell immunity is dependent on the processing and presentation of antigen on MHC class I molecules by professional antigen presenting cells (APC). To develop an improved T cell vaccine for HIV we investigated whether fusing the ubiquitin gene to the N terminus of the HIV gag gene enhanced targeting to the proteasome resulting in better CD8 T cell responses. Human monocyte derived dendritic cells (moDC), transduced with adenovirus vectors carrying either ubiquitinated or non-ubiquitinated gag transgene constructs, were co-cultured with autologous naïve T cells and T cell responses were measured after several weekly cycles of stimulation. Despite targeting of the ubiquitin gag transgene protein to the proteasome, ubiquitination did not increase CD8 T cell immune responses and in some cases diminished responses to gag peptides. There were no marked differences in cytokines produced from ubiquitinated and non-ubiquitinated gag stimulated cultures or in the expression of inhibitory molecules on expanded T cells. However, the ability of moDC transduced with ubiquitinated gag gene to upregulate co-stimulatory molecules was reduced, whilst no difference in moDC maturation was observed with a control ubiquitinated and non-ubiquitinated MART gene. Furthermore moDC transduced with ubiquitinated gag produced more IL-10 than transduction with unmodified gag. Thus failure of gag ubiquitination to enhance CD8 responses may be caused by suppression of moDC maturation. These results indicate that when designing a successful vaccine strategy to target a particular cell population, attention must also be given to the effect of the vaccine on APCs.
Assuntos
Vacinas contra a AIDS/genética , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Ubiquitina/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Vacinas contra a AIDS/imunologia , Animais , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos , Transdução Genética , Transgenes , Ubiquitina/imunologia , Ubiquitinação , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Myeloid dendritic cells (mDCs) are essential for initiation of adaptive immune responses but are depleted in HIV infection. Evidence suggests that apoptosis mediates loss, and to further understand the pathways involved, expression of caspases mediating apoptosis via the extrinsic and intrinsic pathways was analyzed. Blood samples were obtained from 14 HIV-infected patients (nine HAART and five antiretroviral naive) and 10 healthy controls. The expression of intracellular active caspases 8 and 9, associated with extrinsic and intrinsic pathways of apoptosis, and the expression of cell membrane death receptors and their ligands were assessed by flow cytometry in mDC. Additionally, expression of active caspases 8 and 9 in purified mDCs cultured for 5 days with HIV-Bal was analyzed. Frequencies of mDCs in the blood of HIV-infected patients were decreased while expression of CCR7 was up-regulated. Up to 94.4% and 91.8% of mDCs from HIV-infected patients expressed active caspases 8 and 9, respectively, compared to 24.5% and 19.9% from healthy controls (both p<0.0001). However, monocytes and B and T cells from HIV-infected patients also showed increased levels of these caspases. Percentages of FASL expression in mDCs were also elevated in HIV-infected individuals while mDCs expressing cell membrane death receptors remained unchanged. No differences between HAART and naive patients were observed for any of the molecules measured. Caspases 8 and 9 were up-regulated in mDCs cocultured with HIV despite lack of productive infection in vitro. Extrinsic and intrinsic pathways of apoptosis are up-regulated in HIV infection but do not correlate with cell depletion.
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Apoptose/fisiologia , Células Dendríticas/patologia , Células Dendríticas/fisiologia , Infecções por HIV/patologia , Infecções por HIV/fisiopatologia , HIV-1 , Terapia Antirretroviral de Alta Atividade , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/fisiologia , Estudos de Casos e Controles , Caspase 8/metabolismo , Caspase 9/metabolismo , Células Dendríticas/imunologia , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/fisiologia , Receptores CCR7/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/fisiologiaRESUMO
BACKGROUND: High mutation rates of human immunodeficiency virus (HIV) allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition. METHODOLOGY/PRINCIPAL FINDINGS: three vaccines were compared: full-length unmodified SIV-mac239 gag, full-length gag fused at the N-terminus to ubiquitin and 7 gag fragments of equal size spanning the whole of gag with ubiquitin-fused to the N-terminus of each fragment. Genes were cloned into a replication defective adenovirus vector and immunogenicity assessed in an in vitro human priming system. The breadth of the CD8 T cell response, defined by the number of distinct epitopes, was assessed by IFN-γ-ELISPOT and memory phenotype and cytokine production evaluated by flow cytometry. We observed an increase of two- to six-fold in the number of epitopes recognised in the ubiquitin-fused fragments compared to the ubiquitin-fused full-length gag. In contrast, although proteasomal targeting was achieved, there was a marked reduction in the number of epitopes recognised in the ubiquitin-fused full-length gag compared to the full-length unmodified gene, but there were no differences in the number of epitope responses induced by non-ubiquitinated full-length gag and the ubiquitin-fused mini genes. Fragmentation and ubiquitination did not affect T cell memory differentiation and polyfunctionality, though most responses were directed against the Ad5 vector. CONCLUSION/SIGNIFICANCE: Fragmentation but not fusion with ubiquitin increases the breadth of the CD8 T vaccine response against SIV-mac239 gag. Thus gene fragmentation of HIV vaccines may maximise responses.
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Produtos do Gene gag/imunologia , Fragmentos de Peptídeos/imunologia , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T/imunologia , Vacinas Virais/imunologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Produtos do Gene gag/biossíntese , Produtos do Gene gag/genética , Infecções por HIV/prevenção & controle , Humanos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Proteólise , Estabilidade de RNA , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Transdução Genética , Ubiquitinação , Vacinas Virais/biossíntese , Vacinas Virais/genéticaRESUMO
PURPOSE OF REVIEW: Loss of blood plasmacytoid dendritic cell (pDC) in HIV-1 infection is thought to impact on adaptive immune responses whilst the virus also induces aberrant interferon alpha (IFN-α) production that may fuel chronic immune activation and drive disease progression. Recent attention has focussed on the pathway of HIV-induced IFN-α production by pDC and the new data are reviewed here together with the pathway leading to infection. RECENT FINDINGS: Attachment to CD4 and chemokine co-receptors is essential for HIV-1 infection. Although CD4, but not co-receptor binding, is a major route for passage to endosomes and triggering of IFN-α secretion this may also occur by CD4-independent mechanisms involving other receptors. In contrast to other Toll-like receptor (TLR)-7 ligands and RNA viruses that stimulate pDC to secrete IFN-α for 2-3 âh, HIV-1-stimulated pDC can give sustained IFN-α production for up to 48 âh which may contribute to chronic immune activation. This may reflect retention of HIV in early endosomes which also seems to be associated with incomplete maturation induced by HIV. SUMMARY: HIV-1-pDC interactions contribute to pathogenesis through depletion and aberrant IFN-α production. New data on the pathway of pDC HIV-stimulated IFN-α secretion may facilitate therapy to reduce chronic immune activation and slow disease progression.
Assuntos
Células Dendríticas/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Interferon-alfa/biossíntese , Humanos , Interferon-alfa/imunologiaRESUMO
Dendritic cells (DC) are potent inducers of natural killer (NK) cells. There are two distinct populations in blood, myeloid (mDC) and plasmacytoid (pDC) but they can also be generated In vitro from monocytes (mdDC). Although it is established that blood DC are lost in HIV-1 infection, the full impact of HIV-1 infection on DC-NK cell interactions remains elusive. We thus investigated the ability of pDC, mDC, and mdDC from viremic and anti-retroviral therapy-treated aviremic HIV-1+ patients to stimulate various NK cell functions. Stimulated pDC and mdDC from HIV-1+ patients showed reduced secretion of IFN-α and IL-12p70 respectively and their capacity to stimulate expression of CD25 and CD69, and IFN-γ secretion in NK cells was also reduced. pDC activation of NK cell degranulation in response to a tumour cell line was severely reduced in HIV-1+ patients but the ability of mDC to activate NK cells was not affected by HIV-1 infection, with the exception of HLA-DR induction. No differences were observed between viremic and aviremic patients indicating that anti-retroviral therapy had minimal effect on restoration on pDC and mdDC-mediated activation of NK cells. Results from this study provide further insight into HIV-1 mediated suppression of innate immune functions.
Assuntos
Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Monócitos/citologia , Adulto , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Estudos de Coortes , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Feminino , Infecções por HIV/complicações , HIV-1/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/farmacologia , Viremia/complicações , Viremia/imunologia , Adulto JovemRESUMO
Monocyte-derived dendritic cells (MDDCs) have been used in therapeutic vaccination for cancer. A small number of studies have employed a similar approach to vaccinate human immunodeficiency virus (HIV)-infected individuals. We have thus analyzed the functional properties of MDDCs generated from HIV-infected individuals who either are receiving highly active antiretroviral therapy or are therapy naive. There was no difference in the MDDC phenotype or efficiency of MDDC generation between HIV-infected individuals and healthy control subjects. Despite this, the MDDCs derived from both groups of infected individuals were severely impaired in their ability to stimulate the proliferation of allogeneic T cells. Furthermore, production of interferon-gamma was reduced in T cells stimulated by MDDCs. These functional changes may be at least partly explained by reduced interleukin-12 and increased interleukin-10 secretion on stimulation with lipopolysaccharide and CD40 ligand. Our findings suggest that MDDCs used in therapeutic vaccination of HIV-infected individuals may show reduced potency.
Assuntos
Células Dendríticas/fisiologia , Infecções por HIV/metabolismo , HIV-1 , Interleucina-10/biossíntese , Subunidade p40 da Interleucina-12/biossíntese , Linfócitos T/imunologia , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Células Cultivadas , Infecções por HIV/tratamento farmacológico , Humanos , Monócitos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Vaccines against viruses need to target dendritic cells (DC) and stimulate mucosal immunity. Most vaccine studies have focussed on monocyte-derived or dermal DC (dDC) but recent evidence suggests that Langerhans cells (LC) may stimulate mucosal immunity more effectively. New chimeric adenovirus vectors expressing fibre protein from group B adenoviruses (rAd5/11), which utilise CD46 rather than the Coxsackie adenovirus receptor (CAR), have been developed as vaccines to improve transduction and overcome problems of pre-existing vector immunity. Transduction of LC and dDC by rAd5/11 and standard rAd5 expressing green fluorescent protein (GFP) showed that both DC types were more efficiently transduced by rAd5/11 than by rAd5. Although expression of CD46 and the integrins alphavbeta3 and alphavbeta5, which recognise the adenovirus penton base and mediate virus internalisation, was similar in LC and dDC, LC expressed higher levels of GFP. Transduction by electroporation of plasmid also resulted in higher GFP expression in LC, suggesting differences between the two DC populations at a post-entry stage. Transduction with either vector did not induce maturation of LC or dDC and did not affect their ability to stimulate T cells. These findings suggest that vaccine strategies that target LC with adenovirus vectors may be worthy of exploration.