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1.
Oncoimmunology ; 12(1): 2158610, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36545256

RESUMO

Immune-checkpoint inhibitors (ICI) are highly effective in reinvigorating T cells to attack cancer. Nevertheless, a large subset of patients fails to benefit from ICI, partly due to lack of the cancer neoepitopes necessary to trigger an immune response. In this study, we used the thiopurine 6-thioguanine (6TG) to induce random mutations and thus increase the level of neoepitopes presented by tumor cells. Thiopurines are prodrugs which are converted into thioguanine nucleotides that are incorporated into DNA (DNA-TG), where they can induce mutation through single nucleotide mismatching. In a pre-clinical mouse model of a mutation-low melanoma cell line, we demonstrated that 6TG induced clinical-grade DNA-TG integration resulting in an improved tumor control that was strongly T cell dependent. 6TG exposure increased the tumor mutational burden, without affecting tumor cell proliferation and cell death. Moreover, 6TG treatment re-shaped the tumor microenvironment by increasing T and NK immune cells, making the tumors more responsive to immune-checkpoint blockade. We further validated that 6TG exposure improved tumor control in additional mouse models of melanoma. These findings have paved the way for a phase I/II clinical trial that explores whether treatment with thiopurines can increase the proportion of otherwise treatment-resistant cancer patients who may benefit from ICI therapy (NCT05276284).


Assuntos
Melanoma , Tioguanina , Animais , Camundongos , Inibidores de Checkpoint Imunológico , Melanoma/tratamento farmacológico , Melanoma/genética , Mutação , Tioguanina/farmacologia , Tioguanina/uso terapêutico , Microambiente Tumoral , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto
2.
Front Immunol ; 14: 1322536, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38164135

RESUMO

Introduction: Mucosal immunization eliciting local T-cell memory has been suggested for improved protection against respiratory infections caused by viral variants evading pre-existing antibodies. However, it remains unclear whether T-cell targeted vaccines suffice for prevention of viral transmission and to which extent local immunity is important in this context. Methods: To study the impact of T-cell vaccination on the course of viral respiratory infection and in particular the capacity to inhibit viral transmission, we used a mouse model involving natural murine parainfluenza infection with a luciferase encoding virus and an adenovirus based nucleoprotein targeting vaccine. Results and discussion: Prior intranasal immunization inducing strong mucosal CD8+ T cell immunity provided an almost immediate shut-down of the incipient infection and completely inhibited contact based viral spreading. If this first line of defense did not operate, as in parentally immunized mice, recirculating T cells participated in accelerated viral control that reduced the intensity of inter-individual transmission. These observations underscore the importance of pursuing the development of mucosal T-cell inducing vaccines for optimal protection of the individual and inhibition of inter-individual transmission (herd immunity), while at the same time explain why induction of a strong systemic T-cell response may still impact viral transmission.


Assuntos
Linfócitos T CD8-Positivos , Vacinas , Camundongos , Animais , Memória Imunológica , Vacinação , Pulmão
3.
Viruses ; 13(11)2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34835021

RESUMO

It is generally believed that a successful Zika virus (ZIKV) vaccine should induce neutralizing antibodies against the ZIKV envelope (E) protein to efficiently halt viral infection. However, E-specific neutralizing antibodies have been implicated in a phenomenon called antibody-dependent enhancement, which represents an ongoing concern in the flavivirus-vaccinology field. In this report, we investigated the vaccination potential of replication-deficient adenoviral vectors encoding the ZIKV non-structural proteins 1 and 2 (NS1/NS2) and employed the strategy of linking the antigens to the MHC-II associated invariant chain (li) to improve immunogenicity and by inference, the level of protection. We demonstrated that li-linkage enhanced the production of anti-NS1 antibodies and induced an accelerated and prolonged polyfunctional CD8 T cell response in mice, which ultimately resulted in a high degree of protection against ZIKV infection of the CNS.


Assuntos
Antígenos Virais/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/prevenção & controle , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Facilitadores , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Feminino , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos C57BL , Vacinação , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/imunologia , Zika virus/imunologia , Infecção por Zika virus/virologia
4.
Sci Transl Med ; 12(548)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32554708

RESUMO

Strategies to enhance the induction of high magnitude T cell responses through vaccination are urgently needed. Major histocompatibility complex (MHC) class II-associated invariant chain (Ii) plays a critical role in antigen presentation, forming MHC class II peptide complexes for the generation of CD4+ T cell responses. Preclinical studies evaluating the fusion of Ii to antigens encoded in vector delivery systems have shown that this strategy may enhance T cell immune responses to the encoded antigen. We now assess this strategy in humans, using chimpanzee adenovirus 3 and modified vaccinia Ankara vectors encoding human Ii fused to the nonstructural (NS) antigens of hepatitis C virus (HCV) in a heterologous prime/boost regimen. Vaccination was well tolerated and enhanced the peak magnitude, breadth, and proliferative capacity of anti-HCV T cell responses compared to non-Ii vaccines in humans. Very high frequencies of HCV-specific T cells were elicited in humans. Polyfunctional HCV-specific CD8+ and CD4+ responses were induced with up to 30% of CD3+CD8+ cells targeting single HCV epitopes; these were mostly effector memory cells with a high proportion expressing T cell activation and cytolytic markers. No volunteers developed anti-Ii T cell or antibody responses. Using a mouse model and in vitro experiments, we show that Ii fused to NS increases HCV immune responses through enhanced ubiquitination and proteasomal degradation. This strategy could be used to develop more potent HCV vaccines that may contribute to the HCV elimination targets and paves the way for developing class II Ii vaccines against cancer and other infections.


Assuntos
Vacinas Virais , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos T CD8-Positivos , Hepacivirus/genética , Antígenos de Histocompatibilidade Classe II , Humanos
5.
Front Immunol ; 11: 595707, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33603737

RESUMO

Expression of programmed cell death-1 receptor (PD-1) has traditionally been linked to T-cell exhaustion, as signaling via PD-1 dampens the functionality of T-cells upon repetitive antigen exposures during chronic infections. However, resent findings pointing to the involvement of PD-1 both in T-cell survival and in restraining immunopathology, challenge the concept of PD-1 solely as marker for T-cell exhaustion. Tissue resident memory T cells (Trms) hold unique effector qualities, but within a delicate organ like the CNS, these protective abilities could potentially be harmful. In contrast to their counterparts in many other tissues, brain derived CD8+ Trms have been found to uniformly and chronically express PD-1. In this study we utilized a recently established model system for generating CNS Trms in order to improve our understanding regarding the role of PD-1 expression by Trms inside the CNS. By intracerebral (i.c.) inoculation with a non-replicating adeno-viral vector, we induced a PD-1hi CD8+ T cell memory population within the CNS. We found that PD-1 expression lowered the severity of clinical disease associated with the i.c. inoculation. Furthermore, high levels of PD-L1 expression were found on the infiltrating monocytes and macrophages as well as on the resident microglia, oligodendrocytes and astrocytes during the acute phase of the response. Additionally, we showed that the intensity of PD-1 expression correlates with local antigen encounter and found that PD-1 expression was associated with decreased CD8+ T cell memory formation in the CNS despite an increased number of infiltrating CD8+ T cells. Most importantly, our experiments revealed that despite expression of PD-1 and several additional markers linked to T-cell exhaustion, Tim-3, Lag-3 and CD39, the cells did not show signs of limited effector capacity. Collectively, these results endorse the increasing amount of evidence pointing to an immune-modifying role for PD-1 expression within the CNS, a mechanism we found to correlate with local antigen exposure.


Assuntos
Encéfalo/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptor de Morte Celular Programada 1/imunologia , Animais , Antígenos Virais/imunologia , Feminino , Memória Imunológica , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Morte Celular Programada 1/genética
6.
Front Immunol ; 9: 2717, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30534124

RESUMO

Lipocalin-2 is a constituent of the neutrophil secondary granules and is expressed de novo by macrophages and epithelium in response to inflammation. Lipocalin-2 acts in a bacteriostatic fashion by binding iron-loaded siderophores required for bacterial growth. Mycobacterium tuberculosis (M.tb) produces siderophores that can be bound by lipocalin-2. The impact of lipocalin-2 in the innate immune response toward extracellular bacteria has been established whereas the effect on intracellular bacteria, such as M.tb, is less well-described. Here we show that lipocalin-2 surprisingly confers a growth advantage on M.tb in the early stages of infection (3 weeks post-challenge). Using mixed bone marrow chimeras, we demonstrate that lipocalin-2 derived from granulocytes, but not from epithelia and macrophages, leads to increased susceptibility to M.tb infection. In contrast, lipocalin-2 is not observed to promote mycobacterial growth at later stages of M.tb infection. We demonstrate co-localization of granulocytes and mycobacteria within the nascent granulomas at week 3 post-challenge, but not in the consolidated granulomas at week 5. We hypothesize that neutrophil-derived lipocalin-2 acts to supply a source of iron to M.tb in infected macrophages within the immature granuloma, thereby facilitating mycobacterial growth.


Assuntos
Granulócitos/imunologia , Granuloma/imunologia , Imunidade Inata , Lipocalina-2/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Granulócitos/patologia , Granuloma/genética , Granuloma/microbiologia , Granuloma/patologia , Lipocalina-2/genética , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Tuberculose/genética , Tuberculose/patologia
7.
Vaccine ; 36(20): 2825-2832, 2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29627230

RESUMO

Defining correlates of T cell mediated protection is important in order to accelerate the development of efficient T cell based vaccines conferring long-term immunity. Extensive studies have provided important insight regarding the characteristics and functional properties of the effector and memory CD8 T cells induced by viral vector based vaccines. However, long-term protection has been difficult to achieve with T cell inducing vaccines, and the determinants underlying this loss in protection over time are still not fully defined. In this study we analyzed different parameters of the CD8 T cell response as a function of time after vaccination with a human serotype 5 adenovector expressing the glycoprotein (GP) of LCMV tethered to the MHC class II-associated invariant chain. Using this vector we have previously found that CD8 T cells mediate protection from challenge with GP-expressing Listeria monocytogenes at 60 days post vaccination, but only little protection after further 60 days, and we now confirm this observation. A comparison of vaccine-primed CD8 T cells early and late after vaccination revealed a minor decline in the overall numbers of antigen specific memory CD8 T cells during this interval. More importantly, we also observed phenotypic changes over time with a distinct decline in the frequency and number of KLRG1+ CD8 T cells, and, notably, adoptive transfer studies confirmed that memory CD8 T cells expressing KLRG1 are central to protection from systemic L. monocytogenes infection. Together these findings imply that multiple factors including changes in memory T cell numbers and phenotypic composition over time influence the longevity of CD8 T-cell mediated protection.


Assuntos
Adenovírus Humanos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteção Cruzada , Listeria monocytogenes/imunologia , Listeriose/prevenção & controle , Animais , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Fatores de Tempo
8.
EBioMedicine ; 18: 204-215, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28302457

RESUMO

Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat, vif, rev and vpr antigens fused to the MHC class II associated invariant chain. Immunizations induced broad T cell responses in all vaccinees. Following up to 10 repeated low-dose intrarectal challenges, vaccinees suppressed early viral replication (P=0.01) and prevented the peak viremia in 5/6 animals. Despite consistently undetectable viremia in 2 out of 6 vaccinees, all animals showed evidence of infection induced immune responses indicating that infection had taken place. Vaccinees, with and without detectable viremia better preserved their rectal CD4+ T cell population and had reduced immune hyperactivation as measured by naïve T cell depletion, Ki-67 and PD-1 expression on T cells. These results indicate that vaccination towards SIV accessory antigens vaccine can provide a level of acute control of SIV replication with a suggestion of beneficial immunological consequences in infected animals of unknown long-term significance. In conclusion, our studies demonstrate that a vaccine encoding subdominant antigens not normally associated with virus control can exert a significant impact on acute peak viremia.


Assuntos
Antígenos Heterófilos/imunologia , Vetores Genéticos/imunologia , Retrovirus dos Símios/fisiologia , Vacinas contra a SAIDS/imunologia , Adenoviridae/genética , Animais , Antígenos Heterófilos/genética , Antígenos Heterófilos/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Macaca mulatta , Camundongos , Viremia/imunologia , Viremia/prevenção & controle , Replicação Viral/fisiologia
9.
Blood ; 129(7): 866-878, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28003273

RESUMO

Human and mouse chronic lymphocytic leukemia (CLL) develops from CD5+ B cells that in mice and macaques are known to define the distinct B1a B-cell lineage. B1a cells are characterized by lack of germinal center (GC) development, and the B1a cell population is increased in mice with reduced GC formation. As a major mediator of follicular B-cell migration, the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) directs B-cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells toward the extrafollicular area, whereas downregulation is essential for GC formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to CLL. Here, we demonstrate that B-cell-targeted expression of human EBI2 (hEBI2) in mice reduces GC-dependent immune responses, reduces total immunoglobulin M (IgM) and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5+ B1a B-cell subset (present as early as 4 days after birth), late-onset lymphoid cancer development, and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B-cell malignancies.


Assuntos
Linfócitos B/patologia , Centro Germinativo/patologia , Leucemia Linfocítica Crônica de Células B/genética , Linfoma/genética , Receptores Acoplados a Proteínas G/genética , Regulação para Cima , Animais , Linfócitos B/imunologia , Antígenos CD5/análise , Antígenos CD5/imunologia , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/citologia , Centro Germinativo/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma/imunologia , Linfoma/patologia , Camundongos , Receptores Acoplados a Proteínas G/imunologia
11.
Mol Ther ; 22(12): 2107-2117, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25023330

RESUMO

We have previously shown that for the majority of antigens, adenoviral vaccines expressing the target antigen fused to the MHC associated invariant chain (Ii) induce an accelerated, augmented, and prolonged transgene-specific CD8(+) T-cell response. Here we describe a new adenoviral vaccine vector approach where the target antigen fused to Ii is expressed from the adenoviral E1 region and IL-2 is expressed from the E3 region. Immunization of mice with this new vector construct resulted in an augmented primary effector CD8(+) T-cell response. Furthermore, in a melanoma model we observed significantly prolonged tumor control in vaccinated wild type (WT) mice. The improved tumor control required antigen-specific cells, since no tumor control was observed, unless the melanoma cells expressed the vaccine targeted antigen. We also tested our new vaccine in immunodeficient (CD80/86 deficient) mice. Following vaccination with the IL-2 expressing construct, these mice were able to raise a delayed but substantial CD8(+) T-cell response, and to control melanoma growth nearly as efficaciously as similarly vaccinated WT mice. Taken together, these results demonstrate that current vaccine vectors can be improved and even tailored to meet specific demands: in the context of therapeutic vaccination, the capacity to promote an augmented effector T-cell response.


Assuntos
Antígenos Virais de Tumores/genética , Linfócitos T CD8-Positivos/metabolismo , Vetores Genéticos/administração & dosagem , Interleucina-2/genética , Melanoma Experimental/terapia , Neoplasias Cutâneas/terapia , Animais , Antígenos Virais de Tumores/imunologia , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Feminino , Interleucina-2/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Cutâneas/imunologia , Baço/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologia
12.
J Immunol ; 191(8): 4152-64, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24043891

RESUMO

The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC class II-associated invariant chain (Ii) greatly enhances both the presentation of most target Ags, as well as overall protection against viral infection, such as lymphocytic choriomeningitis virus (LCMV). The present study extends this vaccination concept to include protection against intracellular bacteria, using Listeria monocytogenes as a model organism. Protection in C57BL/6 mice against recombinant L. monocytogenes expressing an immunodominant epitope of the LCMV glycoprotein (GP33) was greatly accelerated, augmented, and prolonged following vaccination with an adenoviral vaccine encoding GP linked to Ii compared with vaccination with the unlinked vaccine. Studies using knockout mice demonstrated that CD8(+) T cells were largely responsible for this protection, which is mediated through perforin-dependent lysis of infected cells and IFN-γ production. Taking the concept a step further, vaccination of C57BL/6 (L. monocytogenes-resistant) and BALB/c (L. monocytogenes-susceptible) mice with adenoviral vectors encoding natural L. monocytogenes-derived soluble Ags (listeriolysin O and p60) revealed that tethering of these Ags to Ii markedly improved the vaccine-induced CD8(+) T cell response to two of three epitopes studied. More importantly, Ii linkage accelerated and augmented vaccine-induced protection in both mouse strains and prolonged protection, in particular that induced by the weak Ag, p60, in L. monocytogenes-susceptible BALB/c mice.


Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Listeria monocytogenes/imunologia , Listeriose/prevenção & controle , Adenoviridae/genética , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Vacinas Bacterianas , Sequência de Bases , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos , Glicoproteínas/genética , Glicoproteínas/imunologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Imunidade Celular , Interferon gama/biossíntese , Interferon gama/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Listeriose/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/prevenção & controle , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Antígenos O/genética , Antígenos O/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Perforina/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia
13.
J Virol ; 87(11): 6283-95, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23536658

RESUMO

It has been reported that adenovirus (Ad)-primed CD8 T cells may display a distinct and partially exhausted phenotype. Given the practical implications of this claim, we decided to analyze in detail the quality of Ad-primed CD8 T cells by directly comparing these cells to CD8 T cells induced through infection with lymphocytic choriomeningitis virus (LCMV). We found that localized immunization with intermediate doses of Ad vector induces a moderate number of functional CD8 T cells which qualitatively match those found in LCMV-infected mice. The numbers of these cells may be efficiently increased by additional adenoviral boosting, and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad vaccination led to even higher numbers of memory cells. In this case, the vaccination led to the generation of a population of memory cells characterized by relatively low CD27 expression and high CD127 and killer cell lectin-like receptor subfamily G member 1 (KLRG1) expression. These memory CD8 T cells were capable of proliferating in response to viral challenge and protecting against infection with live virus. Furthermore, viral challenge was followed by sustained expansion of the memory CD8 T-cell population, and the generated memory cells did not appear to have been driven toward exhaustive differentiation. Based on these findings, we suggest that adenovirus-based prime-boost regimens (including Ad serotype 5 [Ad5] and Ad5-like vectors) represent an effective means to induce a substantially expanded, long-lived population of high-quality transgene-specific memory CD8 T cells.


Assuntos
Adenovírus Humanos/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/imunologia , Glicoproteínas/imunologia , Memória Imunológica , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Proteínas Virais/imunologia , Adenovírus Humanos/genética , Animais , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Feminino , Vetores Genéticos/genética , Glicoproteínas/administração & dosagem , Glicoproteínas/genética , Humanos , Coriomeningite Linfocítica/prevenção & controle , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/genética , Camundongos , Camundongos Endogâmicos C57BL , Vacinação , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia
14.
PLoS One ; 7(4): e34884, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22514686

RESUMO

Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.


Assuntos
Adenoviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/imunologia , Adenoviridae/fisiologia , Animais , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL
15.
J Immunol ; 186(7): 3997-4007, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21357263

RESUMO

The impact of prophylactic vaccination against acute and chronic infection in a Th-deficient host has not been adequately addressed because of difficulties in generating protective immunity in the absence of CD4(+) T cell help. In this study, we demonstrated that a broad CD8(+) T cell immune response could be elicited in MHC class II-deficient mice by vaccination with adenovirus encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein tethered to MHC class II-associated invariant chain. Moreover, the response induced conferred significant cytolytic CD8(+) T cell-mediated protection against challenge with a high dose of the invasive clone 13 strain of LCMV. In contrast, vaccination with adenovirus encoding unlinked LCMV glycoprotein induced weak virus control in the absence of CD4(+) T cells, and mice may die of increased immunopathology associated with incomplete protection. Acute mortality was not observed in any vaccinated mice following infection with the less-invasive Traub strain. However, LCMV Traub infection caused accelerated late mortality in unvaccinated MHC class II-deficient mice; in this case, we observed a strong trend toward delayed mortality in vaccinated mice, irrespective of the nature of the vaccine. These results indicated that optimized vaccination may lead to efficient protection against acute viral infection, even in Th-deficient individuals, but that the duration of such immunity is limited. Nevertheless, for select immunodeficiencies in which CD4(+) T cell deficiency is incomplete or transient, these results are very encouraging.


Assuntos
Antígenos de Histocompatibilidade Classe II/genética , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/prevenção & controle , Vírus da Coriomeningite Linfocítica/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Virais/imunologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Doença Crônica , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Epitopos de Linfócito T/genética , Coriomeningite Linfocítica/imunologia , Linfopenia/imunologia , Linfopenia/patologia , Linfopenia/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Auxiliares-Indutores/patologia , Linfócitos T Auxiliares-Indutores/virologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
16.
J Immunol ; 186(4): 2355-64, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21257961

RESUMO

Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8(+) T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8(+) T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8(+) T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.


Assuntos
Adenoviridae/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/imunologia , Hepacivirus/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vacinas contra Hepatite Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Adenoviridae/genética , Animais , Antígenos de Diferenciação de Linfócitos B/administração & dosagem , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Modelos Animais de Doenças , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Hepacivirus/genética , Hepatite C/imunologia , Hepatite C/patologia , Hepatite C/prevenção & controle , Antígenos de Histocompatibilidade Classe II/administração & dosagem , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Memória Imunológica/genética , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Vaccinia virus/genética , Vaccinia virus/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/genética , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/genética
17.
Acta Derm Venereol ; 90(5): 468-73, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20814620

RESUMO

In order to explore the mechanisms of inflammatory skin disorders, we established two methods of expanding skin-derived lymphocytes, one using high levels of interleukin (IL)-2 and IL-4 (method A) and the other using low levels of cytokines and anti-CD3/CD28 microbeads (method B). Both methods provide advantages for functional studies. With either of these two, we could obtain more than 10(7) cells/ from a 3 mm skin biopsy in 21 days from 23 out of 26 biopsies of various skin diseases. The relevance of these cells was confirmed by shifted T-cell receptor beta chain variable region (TCR-Vbeta) repertoire and antigen-dependent proliferation in antigen-driven skin disorders. The propagation of skin-resident lymphocytes, seen especially in method A, seems to be mediated by a functional defect of regulatory T cells residing in skin sequentially expanding under the conditions of our methods.


Assuntos
Anticorpos , Antígenos CD28/imunologia , Complexo CD3/imunologia , Técnicas de Cultura de Células , Proliferação de Células , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Pele/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Biópsia , Linhagem Celular , Separação Celular , Citotoxicidade Imunológica , Dinamarca , Feminino , Citometria de Fluxo , Humanos , Japão , Masculino , Microesferas , Pessoa de Meia-Idade , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas Recombinantes/metabolismo , Pele/patologia , Linfócitos T/patologia , Fatores de Tempo
18.
J Immunol ; 184(8): 4431-9, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20212099

RESUMO

Adenoviral vectors have been widely used for experimental gene therapy and vaccination, yet there is a surprising lack of knowledge connecting the route and dose of adenovirus administration to the induced transgene-specific immune response. We have recently demonstrated polyfunctional CD8(+) T cells and protective memory responses using adenoviral vectors, which seem to contrast with recent reports suggesting that an exhausted CD8(+) T cell phenotype is induced by inoculation with adenoviral vectors. Accordingly, we investigated the route and dose interrelationship for transgene-specific CD8(+) T cells using adenoviral vectors encoding beta-galactosidase applied either s.c. or i.v. Irrespective of the route of inoculation, most of the adenoviral inoculum was found to disseminate systemically as the dose was raised beyond 10(9) particles. The number of transgene-specific CD8(+) T cells correlated positively with dissemination, whereas the functional capacity of the generated T cells correlated inversely with vector dissemination. A comparison of the immune response to s.c. or i.v. administration at moderate doses revealed that inoculation by both routes induced a transient peak of IFN-gamma-producing CD8(+) T cells 2 to 3 wk postinfection, but following i.v. administration, these cells were only detected in the liver. Two to four months after systemic, but not peripheral, immunization, dysfunctional transgene-specific CD8(+) T cells impaired in both cytokine production and important in vivo effector functions, accumulated in the spleen. These findings indicate that the localization of the adenoviral inoculum and not the total Ag load determines the quality of the CD8(+) T cell response induced with adenoviral vaccines.


Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Linfócitos T CD8-Positivos/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Vetores Genéticos/imunologia , Transgenes/imunologia , Carga Viral/imunologia , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/prevenção & controle , Animais , Linfócitos T CD8-Positivos/virologia , Testes Imunológicos de Citotoxicidade/tendências , Relação Dose-Resposta Imunológica , , Vetores Genéticos/administração & dosagem , Vetores Genéticos/normas , Membro Posterior , Memória Imunológica/genética , Injeções Intravenosas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Vaccinia virus/enzimologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Carga Viral/normas , beta-Galactosidase/administração & dosagem , beta-Galactosidase/genética , beta-Galactosidase/imunologia
19.
PLoS One ; 4(4): e5139, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19357780

RESUMO

BACKGROUND: Although CD4 T cells are crucial for defense against M.tb, it is still not clear whether the optimal response against M.tb in fact involves both CD4 and CD8 T cells. To test this, we used a new vaccine strategy that generated a strong balanced T cell response consisting of both CD4 and CD8 T cells. METHODS AND FINDINGS: To compare CD4 and CD8 responses against Ag85B-TB10.4 (H4), H4 was delivered as a subunit vaccine in cationic liposomes (CAF01), expressed in Ad5 (Ad-H4) or as a heterologous prime boost vaccination. H4/CAF01 induced primarily CD4 T cells and Ad-H4 gave predominantly a CD8 T cell response. In contrast, the heterologous prime boost combination resulted in augmentation of both the CD4 and CD8 response. The majority (>40%) of the CD4 T cells induced by the heterologous prime boost protocol were polyfunctional, and expressed IFN-gamma(+), IL-2(+), and TNF-alpha(+), whereas most of the CD8 T cells expressed IFN-gamma(+) and TNF-alpha(+) and possessed strong cytotoxic potential. The heterologous prime boost protocol also gave an increase in protective efficacy against M.tb challenge compared to H4/CAF01 and Ad-H4. Both the H4 specific CD4 and CD8 T cells were recruited to the site of infection, at the onset of infection. However, compared to CD8 T cells, CD4 T cells showed more extensive recruitment and were the main T cell subset proliferating at the site of infection. CONCLUSIONS/SIGNIFICANCE: Heterologous prime boost based on H4, produced an additive effect on the priming of CD4 and CD8 cells and in terms of the protective capacity of the vaccine, and therefore represent an interesting new vaccine strategy against M.tb. However, CD4 and CD8 T cells respond very differently to live M.tb challenge, in a manner which supports the consensus that CD4 T cells do play the major role during the early stages of an M.tb infection.


Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunização Secundária/métodos , Ativação Linfocitária , Mycobacterium tuberculosis/imunologia , Subpopulações de Linfócitos T/imunologia , Vacinas contra a Tuberculose/imunologia , Adjuvantes Imunológicos , Animais , Antígenos de Bactérias/genética , Proliferação de Células , Citocinas/imunologia , Citotoxicidade Imunológica , Feminino , Vetores Genéticos , Humanos , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Fenótipo , Baço/citologia , Baço/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia
20.
APMIS ; 117(5-6): 338-55, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19400861

RESUMO

The host counters a viral infection through a complex response made up of components belonging to both the innate and the adaptive immune system. In this report, we review the mechanisms underlying this response, how it is induced and how it is co-ordinated. As cell-cell communication represents the very essence of immune system physiology, a key to a rapid, efficient and optimally regulated immune response is the ability of the involved cells to rapidly shift between a stationary and a mobile state, combined with stringent regulation of cell migration during the mobile state. Through the co-ordinated recruitment of different cell types intended to work in concert, cellular co-operation is optimized particularly under conditions that may involve rare cells. Consequently, a major focus is placed on presenting an overview of the co-operative events and the associated cell migration, which is essential in mounting an efficient host response and co-ordinating innate and adaptive immunity during a primary viral infection.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Leucócitos/imunologia , Viroses/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Comunicação Celular , Quimiotaxia de Leucócito , Citocinas/fisiologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Imunidade Celular , Células Matadoras Naturais/imunologia , Leucócitos/citologia , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Receptores de Citocinas/fisiologia , Receptores de Reconhecimento de Padrão/fisiologia
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