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Introduction: Despite the recent approval of several therapies in the adjuvant setting of melanoma, tumor relapse still occurs in a significant number of completely resected stage III-IV patients. In this context, the use of cancer vaccines is still relevant and may increase the response to immune checkpoint inhibitors. We previously demonstrated safety, immunogenicity and preliminary evidence of clinical efficacy in stage III/IV resected melanoma patients subjected to a combination therapy based on peptide vaccination together with intermittent low-dose interferon-α2b, with or without dacarbazine preconditioning (https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26). In this setting, we then focused on pre-treatment patient immune status to highlight possible factors associated with clinical outcome. Methods: Multiparametric flow cytometry was used to identify baseline immune profiles in patients' peripheral blood mononuclear cells and correlation with the patient clinical outcome. Receiver operating characteristic curve, Kaplan-Meier survival and principal component analyses were used to evaluate the predictive power of the identified markers. Results: We identified 12 different circulating T and NK cell subsets with significant (p ≤ 0.05) differential baseline levels in patients who later relapsed with respect to patients who remained free of disease. All 12 parameters showed a good prognostic accuracy (AUC>0.7, p ≤ 0.05) and 11 of them significantly predicted the relapse-free survival. Remarkably, 3 classifiers also predicted the overall survival. Focusing on immune cell subsets that can be analyzed through simple surface staining, three subsets were identified, namely regulatory T cells, CD56dimCD16- NK cells and central memory γδ T cells. Each subset showed an AUC>0.8 and principal component analysis significantly grouped relapsing and non-relapsing patients (p=0.034). These three subsets were used to calculate a combination score that was able to perfectly distinguish relapsing and non-relapsing patients (AUC=1; p=0). Noticeably, patients with a combined score ≥2 demonstrated a strong advantage in both relapse-free (p=0.002) and overall (p=0.011) survival as compared to patients with a score <2. Discussion: Predictive markers may be used to guide patient selection for personalized therapies and/or improve follow-up strategies. This study provides preliminary evidence on the identification of peripheral blood immune biomarkers potentially capable of predicting the clinical response to combined vaccine-based adjuvant therapies in melanoma.
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Immunotherapy with immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment providing unprecedented clinical benefits. However, many patients do not respond to ICIs as monotherapy or develop resistance. Combining ICI-based immunotherapy with chemotherapy is a promising strategy to increase response rates, but few rationale-driven chemo-immunotherapy combinations have reached the clinical arena thus far. In the present study, we show that combined anti-PDL1 and anti-PDL2 antibodies optimally synergize with cyclophosphamide but not with cisplatin, and that the magnitude and duration of the therapeutic response is dependent on the immunogenic potential of the drug and of the tumor itself. Hallmarks of successful therapeutic outcomes were the enhanced infiltration by myeloid (mainly cross-presenting dendritic cells, eosinophils, and monocytic myeloid cells) and T lymphocytes into the tumor tissue and the expansion of circulating memory pools. Overall, our results suggest that immunomodulating chemotherapy can be exploited to increase the efficacy of PD1/PDL axis inhibitors in vivo, and that the magnitude of the synergic therapeutic response is affected by tumor-intrinsic immunogenicity.
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Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos , Modelos AnimaisRESUMO
BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.
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Citometria de Fluxo/normas , Imunofenotipagem/normas , Subpopulações de Linfócitos T/classificação , Biomarcadores/sangue , Complexo CD3/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Cor/normas , Citometria de Fluxo/métodos , Humanos , Memória Imunológica , Itália , Antígenos Comuns de Leucócito/sangue , Leucócitos Mononucleares/imunologia , Variações Dependentes do Observador , Receptores CCR7/sangue , Subpopulações de Linfócitos T/imunologiaRESUMO
Clinical studies based on novel rationales and mechanisms of action of chemotherapy agents and cytokines can contribute to the development of new concepts and strategies of antitumor combination therapies. In previous studies, we investigated the paradoxical immunostimulating effects of some chemotherapeutics and the immunoadjuvant activity of interferon alpha (IFN-α) in preclinical and clinical models, thus unraveling novel rationales and mechanisms of action of chemotherapy agents and cytokines for cancer immunotherapy. Here, we carried out a randomized, phase II clinical trial, in which we analyzed the relapse-free (RFS) and overall survival (OS) of 34 completely resected stage III-IV melanoma patients, treated with peptide-based vaccination (Melan-A/MART-1 and NY-ESO-1) in combination with IFN-α2b, with (arm 2) or without (arm 1) dacarbazine preconditioning. All patients were included in the intention-to-treat analysis. At a median follow-up of 4.5 years (interquartile range, 15.4-81.0 months), the rates of RFS were 52.9 and 35.3% in arms 1 and 2, respectively. The 4.5-year OS rates were 68.8% in arm 1 and 62.7% in arm 2. No significant differences were observed between the two arms for both RFS and OS. Interestingly, the RFS and OS curves remained stable starting from 18 and 42 months, respectively. Grade 3 adverse events occurred in 5.9% of patients, whereas grade 4 events were not observed. Both treatments induced a significant expansion of vaccine-specific CD8+ T cells, with no correlation with the clinical outcome. However, treatment-induced increase of polyfunctionality and of interleukin 2 production by Melan-A-specific CD8+ T cells and expansion/activation of natural killer cells correlated with RFS, being observed only in nonrelapsing patients. Despite the recent availability of different therapeutic options, low-cost, low-toxic therapies with long-lasting clinical effects are still needed in patients with high-risk resected stage III/IV melanoma. The combination of peptide vaccination with IFN-α2b showed a minimal toxicity profile and resulted in encouraging RFS and OS rates, justifying further evaluation in clinical trials, which may include the use of checkpoint inhibitors to further expand the antitumor immune response and the clinical outcome. Clinical Trial Registration: https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26.
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The perspective of combining cancer vaccines with immunomodulatory drugs is currently regarded as a highly promising approach for boosting tumor-specific T cell immunity and eradicating residual malignant cells. The efficacy of dendritic cell (DC) vaccination in combination with lenalidomide, an anticancer drug effective in several hematologic malignancies, was investigated in a follicular lymphoma (FL) model. First, we evaluated the in vitro activity of lenalidomide in modulating the immune responses of lymphocytes co-cultured with a new DC subset differentiated with IFN-α (IFN-DC) and loaded with apoptotic lymphoma cells. We next evaluated the efficacy of lenalidomide and IFN-DC-based vaccination, either alone or in combination, in hu-PBL-NOD/SCID mice bearing established human lymphoma. We found that lenalidomide reduced Treg frequency and IL-10 production in vitro, improved the formation of immune synapses of CD8 + lymphocytes with lymphoma cells and enhanced anti-lymphoma cytotoxicity. Treatment of lymphoma-bearing mice with either IFN-DC vaccination or lenalidomide led to a significant decrease in tumor growth and lymphoma cell spread. Lenalidomide treatment was shown to substantially inhibit tumor-induced neo-angiogenesis rather than to exert a direct cytotoxic effect on lymphoma cells. Notably, the combined treatment with the vaccine plus lenalidomide was more effective than either single treatment, resulting in the significant regression of established tumors and delayed tumor regrowth upon treatment discontinuation. In conclusion, our data demonstrate that IFN-DC-based vaccination plus lenalidomide exert an additive therapeutic effect in xenochimeric mice bearing established lymphoma. These results may pave the way to evaluate this combination in the clinical ground.
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Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Sinergismo Farmacológico , Fatores Imunológicos/imunologia , Interferon-alfa/imunologia , Lenalidomida/farmacologia , Linfoma Folicular/terapia , Animais , Terapia Combinada , Feminino , Humanos , Fatores Imunológicos/farmacologia , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The alarmin IL-33 is an IL-1 family member that stimulates pleiotropic immune reactions depending on the target tissue and microenvironmental factors. In this study, we have investigated the role of IL-33/ST2 axis in antitumor response to melanoma. Injection of IL-33 in mice-bearing subcutaneous B16.F10 melanoma resulted in significant tumor growth delay. This effect was associated with intratumoral accumulation of CD8+ T cells and eosinophils, decrease of immunosuppressive myeloid cells, and a mixed Th1/Th2 cytokine expression pattern with local and systemic activation of CD8+ T and NK cells. Moreover, intranasal administration of IL-33 determined ST2-dependent eosinophil recruitment in the lung that prevented the onset of pulmonary metastasis after intravenous injection of melanoma cells. Accordingly, ST2-deficient mice developed pulmonary metastasis at higher extent than wild-type counterparts, associated with lower eosinophil frequencies in the lung. Of note, depletion of eosinophils by in vivo treatment with anti-Siglec-F antibody abolished the ability of IL-33 to both restrict primary tumor growth and metastasis formation. Finally, we show that IL-33 is able to activate eosinophils resulting in efficient killing of target melanoma cells, suggesting a direct antitumor activity of eosinophils following IL-33 treatment. Our results advocate for an eosinophil-mediated antitumoral function of IL-33 against melanoma, thus opening perspectives for novel cancer immunotherapy strategies.
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Resistance to IFN-I-induced antineoplastic effects has been reported in many tumors and arises, in part, from epigenetic silencing of IFN-stimulated genes by DNA methylation. We hypothesized that restoration of IFN-stimulated genes by co-administration of the demethylating drug 5-aza-2'-deoxycitidine (decitabine [DAC]) may enhance the susceptibility to IFN-I-mediated antitumoral effects in melanoma. We show that combined administration of IFN-I and DAC significantly inhibits the growth of murine and human melanoma cells, both in vitro and in vivo. Compared with controls, DAC/IFN-I-treated melanoma cells exhibited reduced cell growth, augmented apoptosis, and diminished migration. Moreover, IFN-I and DAC synergized to suppress the growth of three-dimensional human melanoma spheroids, altering tumor architecture. These direct antitumor effects correlated with induction of the IFN-stimulated gene Mx1. In vivo, DAC/IFN-I significantly reduced melanoma growth via stimulation of adaptive immunity, promoting tumor-infiltrating CD8+ T cells while inhibiting the homing of immunosuppressive CD11b+ myeloid cells and regulatory T cells. Accordingly, exposure of human melanoma cells to DAC/IFN-I induced the recruitment of immune cells toward the tumor in a Matrigel (Corning Life Sciences, Kennebunkport, ME)-based microfluidic device. Our findings underscore a beneficial effect of DAC plus IFN-I combined treatment against melanoma through both direct and immune-mediated anti-tumor effects.
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Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Interferon Tipo I/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Análise de Variância , Animais , Apoptose/genética , Azacitidina/análogos & derivados , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Humanos , Interferon Tipo I/genética , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Cutâneas/patologia , Estatísticas não ParamétricasRESUMO
BACKGROUND: Advanced melanoma patients have an extremely poor long term prognosis and are in strong need of new therapies. The recently developed targeted therapies have resulted in a marked antitumor effect, but most responses are partial and some degree of toxicity remain the major concerns. Dendritic cells play a key role in the activation of the immune system and have been typically used as ex vivo antigen-loaded cell drugs for cancer immunotherapy. Another approach consists in intratumoral injection of unloaded DCs that can exploit the uptake of a wider array of tumor-specific and individual unique antigens. However, intratumoral immunization requires DCs endowed at the same time with properties typically belonging to both immature and mature DCs (i.e. antigen uptake and T cell priming). DCs generated in presence of interferon-alpha (IFN-DCs), due to their features of partially mature DCs, capable of efficiently up-taking, processing and cross-presenting antigens to T cells, could successfully carry out this task. Combining intratumoral immunization with tumor-destructing therapies can induce antigen release in situ, facilitating the injected DCs in triggering an antitumor immune response. METHODS: We tested in a phase I clinical study in advanced melanoma a chemo-immunotherapy approach based on unloaded IFN-DCs injected intratumorally one day after administration of dacarbazine. Primary endpoint of the study was treatment safety and tolerability. Secondary endpoints were immune and clinical responses of patients. RESULTS: Six patients were enrolled, and only three completed the treatment. The chemo-immunotherapy was well tolerated with no major side effects. Three patients showed temporary disease stabilization and two of them showed induction of T cells specific for tyrosinase, NY-ESO-1 and gp100. Of interest, one patient showing a remarkable long-term disease stabilization kept showing presence of tyrosinase specific T cells in PBMC and high infiltration of memory T cells in the tumor lesion at 21 months. CONCLUSION: We tested a chemo-immunotherapeutic approach based on IFN-DCs injected intratumorally one day after DTIC in advanced melanoma. The treatment was well tolerated, and clinical and immunological responses, including development of vitiligo, were observed, therefore warranting additional clinical studies aimed at evaluating efficacy of this approach. TRIAL REGISTRATION: Trial Registration Number not publicly available due to EudraCT regulations: https://www.clinicaltrialsregister.eu/doc/EU_CTR_FAQ.pdf.
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Dacarbazina/química , Células Dendríticas/citologia , Tratamento Farmacológico/métodos , Imunoterapia/métodos , Injeções Intralesionais , Interferon-alfa/metabolismo , Melanoma/terapia , Adulto , Idoso , Antígenos de Neoplasias/metabolismo , Vacinas Anticâncer/imunologia , Terapia Combinada/métodos , Feminino , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/citologia , Masculino , Proteínas de Membrana/metabolismo , Microscopia Confocal , Pessoa de Meia-Idade , Monócitos/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Vitiligo/induzido quimicamente , Antígeno gp100 de Melanoma/metabolismoRESUMO
The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.
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Antígenos de Neoplasias/uso terapêutico , Biomarcadores Tumorais/imunologia , Vacinas Anticâncer/imunologia , Citometria de Fluxo/métodos , Melanoma/terapia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Melanoma/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.
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Células Dendríticas/virologia , Anticorpos Anti-HIV/metabolismo , HIV-1/metabolismo , Integrinas/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação , Células Dendríticas/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Integrinas/imunologia , Macaca fascicularis , Masculino , Simulação de Acoplamento Molecular , Testes de Neutralização , Oligopeptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Internalização do Vírus , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Certain chemotherapeutic drugs, such as cyclophosphamide (CTX), can enhance the antitumor efficacy of immunotherapy because of their capacity to modulate innate and adaptive immunity. Indeed, it has been argued that this capacity may be more significant to chemotherapeutic efficacy in general than is currently appreciated. To gain insights into the core mechanisms of chemoimmunotherapy, we methodically profiled the effects of CTX on gene expression in bone marrow, spleen, and peripheral blood, and on cytokine expression in plasma and bone marrow of tumor-bearing mice. Gene and protein expression were modulated early and transiently by CTX, leading to upregulation of a variety of immunomodulatory factors, including danger signals, pattern recognition receptors, inflammatory mediators, growth factors, cytokines, chemokines, and chemokine receptors. These factors are involved in sensing CTX myelotoxicity and activating repair mechanisms, which, in turn, stimulate immunoactivation events that promote efficacy. In particular, CTX induced a T-helper 17 (Th17)-related gene signature associated with an increase in Th17, Th1, and activated CD25(+)CD4(+)Foxp3(-) T lymphocytes and a slight recovery of regulatory T cells. By analyzing gene and protein expression kinetics and their relationship to the antitumor efficacy of different therapeutic schedules of combination, we determined that optimal timing for performing adoptive immunotherapy is approximately 1 day after CTX treatment. Together, our findings highlight factors that may propel the efficacy of chemoimmunotherapy, offering a mechanistic glimpse of the important immune modulatory effects of CTX.
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Ciclofosfamida/farmacologia , Tratamento Farmacológico/métodos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Imunoterapia/métodos , Neoplasias/terapia , Animais , Antineoplásicos Alquilantes/farmacologia , Terapia Combinada/métodos , Sistema Imunitário , Camundongos , Camundongos Endogâmicos DBA , Metástase Neoplásica , Neoplasias/genética , Células Th1/citologia , Células Th17/metabolismoRESUMO
Nonintegrating lentiviral vectors are being developed as a efficient and safe delivery system for both gene therapy and vaccine purposes. Several reports have demonstrated that a single immunization with integration-defective lentiviral vectors (IDLVs) delivering viral or tumor model antigens in mice was able to elicit broad and long-lasting specific immune responses in the absence of vector integration. At present, no evidence has been reported showing that IDLVs are able to expand preexisting immune responses in the human context. In the present study, we demonstrate that infection of human antigen-presenting cells (APCs), such as monocyte-derived dendritic cells (DCs) and macrophages with IDLVs expressing influenza matrix M1 protein resulted in effective induction of in vitro expansion of M1-primed CD8(+) T cells, as evaluated by both pentamer staining and cytokine production. This is the first demonstration that IDLVs represent an efficient delivery system for gene transfer and expression in human APCs, useful for immunotherapeutic applications.
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Células Apresentadoras de Antígenos/virologia , Linfócitos T CD8-Positivos/virologia , Vetores Genéticos , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Terapia Genética , Humanos , Imunização , Integrases/genética , Integrases/imunologia , Lentivirus/genética , Lentivirus/imunologia , Macrófagos/imunologia , Monócitos/citologia , Monócitos/imunologia , Transdução Genética , Proteínas Virais/imunologiaRESUMO
We previously reported that cynomolgus monkeys vaccinated with the human immunodeficiency virus (HIV)-1 Tat protein controlled infection after challenge with the simian human immunodeficiency virus (SHIV) 89.6P(cy243) for up to 2 y of follow-up. To evaluate the breadth of the protective immunity elicited by the Tat protein, the vaccines along with the naïve monkeys were intravenously rechallenged with a fivefold higher dose (50 MID(50)) of the same SHIV-89.6P(cy243). The vaccinated monkeys exhibited a statistically significant and long-lasting reduction of viral replication compared to control monkeys. This effect was associated with a strong anamnestic response to Tat, while responses to Gag and Env were nearly undetectable. Taken together, these data provide further evidence for the usefulness of Tat-based vaccines.
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Vacinas contra a AIDS/imunologia , HIV/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia , Animais , Contagem de Linfócito CD4 , Proliferação de Células , HIV/genética , Anticorpos Anti-HIV/sangue , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Macaca fascicularis , Recombinação Genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/genética , Carga ViralRESUMO
Tat is an early regulatory protein that plays a major role in human HIV-1 replication and AIDS pathogenesis, and therefore, it represents a key target for the host immune response. In natural infection, however, Abs against Tat are produced only by a small fraction (approximately 20%) of asymptomatic individuals and are rarely seen in progressors, suggesting that Tat may possess properties diverting the adaptive immunity from generating humoral responses. Here we show that a Th1-type T cell response against Tat is predominant over a Th2-type B cell response in natural HIV-1 infection. This is likely due to the capability of Tat to selectively target and very efficiently enter CD1a-expressing monocyte-derived dendritic cells (MDDC), which represent a primary target for the recognition and response to virus Ag. Upon cellular uptake, Tat induces MDDC maturation and Th1-associated cytokines and beta-chemokines production and polarizes the immune response in vitro to the Th1 pattern through the transcriptional activation of TNF-alpha gene expression. This requires the full conservation of Tat transactivation activity since neither MDDC maturation nor TNF-alpha production are found with either an oxidized Tat, which does not enter MDDC, or with a Tat protein mutated in the cysteine-rich region (cys22 Tat), which enters MDDC as the wild-type Tat but is transactivation silent. Consistently with these data, inoculation of monkeys with the native wild-type Tat induced a predominant Th1 response, whereas cys22 Tat generated mostly Th2 responses, therefore providing evidence that Tat induces a predominant Th1 polarized adaptive immune response in the host.
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Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , HIV-1/imunologia , Células Th1/imunologia , Células Th1/virologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/fisiologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Adulto , Idoso , Animais , Células Cultivadas , Quimiocinas CC/biossíntese , Citocinas/biossíntese , Células Dendríticas/metabolismo , Feminino , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/prevenção & controle , Células HeLa , Humanos , Imunidade Inata , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/virologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Células Th1/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Infection of Macaca fascicularis (cynomolgus monkey) with chimeric simian/human immunodeficiency virus (SHIV) provides a valuable experimental animal model of AIDS and is widely used for the development of human immunodeficiency virus vaccine strategies. In these settings, analysis of CD8(+) T-cell responses during infection represents one of the key parameters for monitoring the evaluation of containment of virus replication. The generation of Gag-specific CD8(+) T cells was reported previously from a cynomolgus monkey infected with SHIV89.6P by taking advantage of a B-lymphoblastoid cell line transduced with a retroviral vector expressing simian immunodeficiency virus (SIV) Gag. Here, it was shown that these cytotoxic T lymphocytes (CTLs) demonstrated specificity for a single 9 aa peptide (NCVGDHQAA) spanning aa 192-200 of the SIVmac239 p55(gag) protein. Furthermore, a positive response was found against the same epitope in one of six other SHIV-infected monkeys. This newly identified SIV Gag CTL epitope in SHIV-infected cynomolgus monkeys will be a useful tool for monitoring and evaluating Gag-specific immune responses during vaccination and infection in the cynomolgus monkey model of AIDS.
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Epitopos de Linfócito T/imunologia , HIV , Vírus Reordenados/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Citotoxicidade Imunológica , Produtos do Gene gag/imunologia , HIV/genética , Interferon gama/biossíntese , Leucócitos Mononucleares/imunologia , Macaca fascicularis , Peptídeos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Especificidade da EspécieRESUMO
Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.