29 results on '"Nájera JL"'
Search Results
2. NYVAC vector modified by C7L viral gene insertion improves T cell immune responses and effectiveness against leishmaniasis.
- Author
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Sánchez-Sampedro L, Mejías-Pérez E, S Sorzano CÓ, Nájera JL, and Esteban M
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- Animals, Antigens, Protozoan genetics, Antigens, Protozoan immunology, Disease Models, Animal, Female, Gene Expression, Genetic Vectors chemistry, HeLa Cells, Humans, Immunization, Secondary, Interferon-gamma genetics, Interferon-gamma immunology, Interleukin-2 genetics, Interleukin-2 immunology, Leishmania major genetics, Leishmania major immunology, Leishmaniasis Vaccines biosynthesis, Leishmaniasis Vaccines genetics, Leishmaniasis, Cutaneous immunology, Leishmaniasis, Cutaneous parasitology, Lysosomal Membrane Proteins genetics, Lysosomal Membrane Proteins immunology, Mice, Mice, Inbred BALB C, Protozoan Proteins genetics, Protozoan Proteins immunology, T-Lymphocytes immunology, T-Lymphocytes parasitology, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha immunology, Vaccinia virus genetics, Vaccinia virus immunology, Viral Proteins immunology, Virus Replication, Genetic Vectors immunology, Leishmania major drug effects, Leishmaniasis Vaccines administration & dosage, Leishmaniasis, Cutaneous prevention & control, T-Lymphocytes drug effects, Viral Proteins genetics
- Abstract
The NYVAC poxvirus vector is used as vaccine candidate for HIV and other diseases, although there is only limited experimental information on its immunogenicity and effectiveness for use against human pathogens. Here we defined the selective advantage of NYVAC vectors in a mouse model by comparing the immune responses and protection induced by vectors that express the LACK (Leishmania-activated C-kinase antigen), alone or with insertion of the viral host range gene C7L that allows the virus to replicate in human cells. Using DNA prime/virus boost protocols, we show that replication-competent NYVAC-LACK that expresses C7L (NYVAC-LACK-C7L) induced higher-magnitude polyfunctional CD8(+) and CD4(+) primary adaptive and effector memory T cell responses (IFNγ, TNFα, IL-2, CD107a) to LACK antigen than non-replicating NYVAC-LACK. Compared to NYVAC-LACK, the NYVAC-LACK-C7L-induced CD8(+) T cell population also showed higher proliferation when stimulated with LACK antigen. After a challenge by subcutaneous Leishmania major metacyclic promastigotes, NYVAC-LACK-C7L-vaccinated mouse groups showed greater protection than the NYVAC-LACK-vaccinated group. Our results indicate that the type and potency of immune responses induced by LACK-expressing NYVAC vectors is improved by insertion of the C7L gene, and that a replication-competent vector as a vaccine renders greater protection against a human pathogen than a non-replicating vector., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
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3. Suppression of NYVAC Infection in HeLa Cells Requires RNase L but Is Independent of Protein Kinase R Activity.
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Fernández-Escobar M, Nájera JL, Baldanta S, Rodriguez D, Way M, Esteban M, and Guerra S
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- HeLa Cells, Humans, Protein Biosynthesis, Endoribonucleases metabolism, Epithelial Cells immunology, Host-Pathogen Interactions, Membrane Glycoproteins antagonists & inhibitors, Vaccinia virus immunology, Viral Envelope Proteins antagonists & inhibitors, Viral Proteins antagonists & inhibitors, eIF-2 Kinase metabolism
- Abstract
Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viral A27L and B5R gene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activity., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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4. NFκB activation by modified vaccinia virus as a novel strategy to enhance neutrophil migration and HIV-specific T-cell responses.
- Author
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Di Pilato M, Mejías-Pérez E, Zonca M, Perdiguero B, Gómez CE, Trakala M, Nieto J, Nájera JL, Sorzano CO, Combadière C, Pantaleo G, Planelles L, and Esteban M
- Subjects
- Animals, Antigen-Presenting Cells immunology, Cell Line, Gene Deletion, Genes, Viral, HIV Antigens immunology, Humans, Lymphocyte Activation immunology, Mice, Inbred BALB C, Mice, Inbred C57BL, Models, Biological, Neutrophil Infiltration, Species Specificity, Vaccinia immunology, Vaccinia virology, Vaccinia virus genetics, CD8-Positive T-Lymphocytes immunology, HIV-1 immunology, Immune System Diseases, Leukocyte Disorders, NF-kappa B metabolism
- Abstract
Neutrophils are antigen-transporting cells that generate vaccinia virus (VACV)-specific T-cell responses, yet how VACV modulates neutrophil recruitment and its significance in the immune response are unknown. We generated an attenuated VACV strain that expresses HIV-1 clade C antigens but lacks three specific viral genes (A52R, K7R, and B15R). We found that these genes act together to inhibit the NFκB signaling pathway. Triple ablation in modified virus restored NFκB function in macrophages. After virus infection of mice, NFκB pathway activation led to expression of several cytokines/chemokines that increased the migration of neutrophil populations (Nα and Nβ) to the infection site. Nβ cells displayed features of antigen-presenting cells and activated virus-specific CD8 T cells. Enhanced neutrophil trafficking to the infection site correlated with an increased T-cell response to HIV vector-delivered antigens. These results identify a mechanism for poxvirus-induced immune response and alternatives for vaccine vector design.
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- 2015
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5. Evaluation of a commercial vaccine against avian poxvirus in turkeys kept in the backyard system in the state of Yucatan, Mexico.
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Estrella-Tec JE, Gutiérrez-Ruiz EJ, Ramírez-González S, Aranda-Cirerol F, Santos-Ricalde R, and Puerto-Nájera JL
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- Animals, Incidence, Mexico epidemiology, Poxviridae Infections epidemiology, Poxviridae Infections prevention & control, Risk Assessment, Avipoxvirus immunology, Poultry Diseases epidemiology, Poultry Diseases prevention & control, Poultry Diseases virology, Poxviridae Infections veterinary, Turkeys, Viral Vaccines pharmacology
- Abstract
One hundred and sixty 1-month-old turkey poults were delivered to 40 households in four communities of the State of Yucatan, Mexico. The poults were divided into two populations, one vaccinated and the other non-vaccinated against avian pox. During three months, monthly visits were carried out in order to monitor the appearance of lesions suggesting avian pox in the birds delivered. Each turkey was clinically examined, searching for characteristic avian pox lesions that were classified according to the degree of severity observed. The true incidence rate and the cumulative incidence rate of avian pox were determined and the true incidence and cumulative incidence rates of mortality were determined and the relative risks calculated. The true incidence rates for avian pox in vaccinated and non-vaccinated birds were 1.5 and 1.47 respectively. The cumulative incidence rates were 0.94 and 0.90 for vaccinated and non-vaccinated birds, respectively. The comparison for the whole period between vaccinated and non-vaccinated groups did not show a significant statistical difference for mortality. However, when mortality was compared between vaccinated and non-vaccinated turkeys for each month of the study, there was a statistically significant difference for the first month (relative risk = 0.216, confidence interval 0.069 to 0.676). In addition, when the severity of pox lesions between groups was compared, statistically significant differences were found in favour of the vaccinated birds (P < 0.0001).
- Published
- 2013
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6. Removal of vaccinia virus genes that block interferon type I and II pathways improves adaptive and memory responses of the HIV/AIDS vaccine candidate NYVAC-C in mice.
- Author
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Gómez CE, Perdiguero B, Nájera JL, Sorzano CO, Jiménez V, González-Sanz R, and Esteban M
- Subjects
- AIDS Vaccines genetics, Animals, CD8-Positive T-Lymphocytes immunology, Chick Embryo, Gene Deletion, Genetic Vectors immunology, HIV Envelope Protein gp120 genetics, HIV Envelope Protein gp120 immunology, HIV Infections immunology, HIV Infections prevention & control, HIV-1 genetics, HIV-1 immunology, Haplorhini, Mice, Mice, Inbred BALB C, Signal Transduction immunology, T-Lymphocytes immunology, Vaccinia virus genetics, Vaccinia virus immunology, AIDS Vaccines immunology, Adaptive Immunity, Immunologic Memory, Interferon Type I metabolism, Interferon-gamma metabolism
- Abstract
Poxviruses encode multiple inhibitors of the interferon (IFN) system, acting at different levels and blocking the induction of host defense mechanisms. Two viral gene products, B19 and B8, have been shown to act as decoy receptors of type I and type II IFNs, blocking the binding of IFN to its receptor. Since IFN plays a major role in innate immune responses, in this investigation we asked to what extent the viral inhibitors of the IFN system impact the capacity of poxvirus vectors to activate immune responses. This was tested in a mouse model with single and double deletion mutants of the vaccine candidate NYVAC-C, which expresses the HIV-1 Env, Gag, Pol, and Nef antigens. When deleted individually or in double, the type I (B19) and type II (B8) IFN binding proteins were not required for virus replication in cultured cells. Studies of immune responses in mice after DNA prime/NYVAC boost revealed that deletion of B8R and/or B19R genes improved the magnitude and quality of HIV-1-specific CD8(+) T cell adaptive immune responses and impacted their memory phase, changing the contraction, the memory differentiation, the effect magnitude, and the functionality profile. For B cell responses, deletion of the viral gene B8R and/or B19R had no effect on antibody levels to HIV-1 Env. These findings revealed that single or double deletion of viral factors (B8 and B19) targeting the IFN pathway is a useful approach in the design of improved poxvirus-based vaccines.
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- 2012
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7. Deletion of the viral anti-apoptotic gene F1L in the HIV/AIDS vaccine candidate MVA-C enhances immune responses against HIV-1 antigens.
- Author
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Perdiguero B, Gómez CE, Nájera JL, Sorzano CO, Delaloye J, González-Sanz R, Jiménez V, Roger T, Calandra T, Pantaleo G, and Esteban M
- Subjects
- Amino Acid Sequence, Animals, Apoptosis genetics, Apoptosis immunology, Base Sequence, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes metabolism, Cell Line, Chick Embryo, Cytokines metabolism, Dendritic Cells immunology, Dendritic Cells metabolism, Epitopes, T-Lymphocyte immunology, Gene Deletion, Gene Expression, Gene Order, Genetic Vectors genetics, Genetic Vectors immunology, HIV Antigens genetics, HIV Envelope Protein gp120 chemistry, HIV Envelope Protein gp120 genetics, HIV Envelope Protein gp120 immunology, HIV-1 genetics, Human Immunodeficiency Virus Proteins chemistry, Human Immunodeficiency Virus Proteins genetics, Human Immunodeficiency Virus Proteins immunology, Humans, Immunologic Memory immunology, Interferon Type I metabolism, Macrophages immunology, Macrophages metabolism, Mice, Molecular Sequence Data, T-Lymphocytes immunology, T-Lymphocytes metabolism, AIDS Vaccines immunology, HIV Antigens immunology, HIV-1 immunology, Vaccinia virus genetics, Vaccinia virus immunology, Viral Proteins genetics, Viral Proteins immunology
- Abstract
Vaccinia virus (VACV) encodes an anti-apoptotic Bcl-2-like protein F1 that acts as an inhibitor of caspase-9 and of the Bak/Bax checkpoint but the role of this gene in immune responses is not known. Because dendritic cells that have phagocytosed apoptotic infected cells cross-present viral antigens to cytotoxic T cells inducing an antigen-specific immunity, we hypothesized that deletion of the viral anti-apoptotic F1L gene might have a profound effect on the capacity of poxvirus vectors to activate specific immune responses to virus-expressed recombinant antigens. This has been tested in a mouse model with an F1L deletion mutant of the HIV/AIDS vaccine candidate MVA-C that expresses Env and Gag-Pol-Nef antigens (MVA-C-ΔF1L). The viral gene F1L is not required for virus replication in cultured cells and its deletion in MVA-C induces extensive apoptosis and expression of immunomodulatory genes in infected cells. Analysis of the immune responses induced in BALB/c mice after DNA prime/MVA boost revealed that, in comparison with parental MVA-C, the mutant MVA-C-ΔF1L improves the magnitude of the HIV-1-specific CD8 T cell adaptive immune responses and impacts on the CD8 T cell memory phase by enhancing the magnitude of the response, reducing the contraction phase and changing the memory differentiation pattern. These findings reveal the immunomodulatory role of F1L and that the loss of this gene is a valid strategy for the optimization of MVA as vaccine vector.
- Published
- 2012
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8. The HIV/AIDS vaccine candidate MVA-B administered as a single immunogen in humans triggers robust, polyfunctional, and selective effector memory T cell responses to HIV-1 antigens.
- Author
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Gómez CE, Nájera JL, Perdiguero B, García-Arriaza J, Sorzano CO, Jiménez V, González-Sanz R, Jiménez JL, Muñoz-Fernández MA, López Bernaldo de Quirós JC, Guardo AC, García F, Gatell JM, Plana M, and Esteban M
- Subjects
- AIDS Vaccines genetics, Drug Carriers, Genetic Vectors, HIV Antigens genetics, HIV-1 immunology, Humans, Immunization, Secondary methods, Injections, Intramuscular, Interferon-gamma biosynthesis, Spain, Time Factors, Vaccination methods, Vaccinia virus genetics, Vaccinia virus immunology, AIDS Vaccines administration & dosage, AIDS Vaccines immunology, Acquired Immunodeficiency Syndrome prevention & control, HIV Antigens immunology, Immunologic Memory, T-Lymphocytes immunology
- Abstract
Attenuated poxvirus vectors expressing human immunodeficiency virus type 1 (HIV-1) antigens are considered promising HIV/AIDS vaccine candidates. Here, we describe the nature of T cell immune responses induced in healthy volunteers participating in a phase I clinical trial in Spain after intramuscular administration of three doses of the recombinant MVA-B-expressing monomeric gp120 and the fused Gag-Pol-Nef (GPN) polyprotein of clade B. The majority (92.3%) of the volunteers immunized had a positive specific T cell response at any time postvaccination as detected by gamma interferon (IFN-γ) intracellular cytokine staining (ICS) assay. The CD4(+) T cell responses were predominantly Env directed, whereas the CD8(+) T cell responses were similarly distributed against Env, Gag, and GPN. The proportion of responders after two doses of MVA-B was similar to that obtained after the third dose of MVA-B vaccination, and the responses were sustained (84.6% at week 48). Vaccine-induced CD8(+) T cells to HIV-1 antigens after 1 year were polyfunctional and distributed mainly within the effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cell populations. Antivector T cell responses were mostly induced by CD8(+) T cells, highly polyfunctional, and of TEMRA phenotype. These findings demonstrate that the poxvirus MVA-B vaccine candidate given alone is highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4 and CD8 T cell responses to HIV-1 antigens, with preference for TEM. Thus, on the basis of the immune profile of MVA-B in humans, this immunogen can be considered a promising HIV/AIDS vaccine candidate.
- Published
- 2011
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9. Safety and immunogenicity of a modified pox vector-based HIV/AIDS vaccine candidate expressing Env, Gag, Pol and Nef proteins of HIV-1 subtype B (MVA-B) in healthy HIV-1-uninfected volunteers: A phase I clinical trial (RISVAC02).
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García F, Bernaldo de Quirós JC, Gómez CE, Perdiguero B, Nájera JL, Jiménez V, García-Arriaza J, Guardo AC, Pérez I, Díaz-Brito V, Conde MS, González N, Alvarez A, Alcamí J, Jiménez JL, Pich J, Arnaiz JA, Maleno MJ, León A, Muñoz-Fernández MA, Liljeström P, Weber J, Pantaleo G, Gatell JM, Plana M, and Esteban M
- Subjects
- AIDS Vaccines administration & dosage, AIDS Vaccines genetics, Adolescent, Adult, Cytokines metabolism, Double-Blind Method, Enzyme-Linked Immunospot Assay, Female, HIV Antibodies blood, HIV-1 genetics, Human Experimentation, Humans, Injections, Intramuscular, Leukocytes, Mononuclear immunology, Male, Middle Aged, Placebos administration & dosage, Vaccines, Synthetic administration & dosage, Vaccines, Synthetic genetics, Vaccines, Synthetic immunology, Viral Proteins genetics, Young Adult, AIDS Vaccines adverse effects, AIDS Vaccines immunology, Drug Carriers, Genetic Vectors, HIV-1 immunology, Vaccinia virus genetics, Viral Proteins immunology
- Abstract
Background: To investigate the safety and immunogenicity of a modified vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B), a phase-I, doubled-blind placebo-controlled trial was performed., Methods: 30 HIV-uninfected volunteers at low risk of HIV-1 infection were randomly allocated to receive 3 intramuscular injections (1×10(8)pfu/dose) of MVA-B (n=24) or placebo (n=6) at weeks 0, 4 and 16. All volunteers were followed 48 weeks. Primary end-points were adverse events and immunogenicity., Results: A total of 169 adverse events were reported, 164 of grade 1-2, and 5 of grade 3 (none related to vaccination). Overall 75% of the volunteers showed positive ELISPOT responses at any time point. The magnitude (median) of the total responses induced was 288SFC/10(6)PBMC at week 18. Antibody responses against Env were observed in 95% and 72% of vaccinees at week 18 and 48, respectively. HIV-1 neutralizing antibodies were detected in 33% of volunteers., Conclusions: MVA-B was safe, well tolerated and elicited strong and durable T-cell and antibody responses in 75% and 95% of volunteers, respectively. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Clinical Trials.gov identifier: NCT00679497., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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10. MVA and NYVAC as vaccines against emergent infectious diseases and cancer.
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Gómez CE, Nájera JL, Krupa M, Perdiguero B, and Esteban M
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- Animals, Cancer Vaccines genetics, Genetic Vectors genetics, Humans, Vaccines, DNA, Vaccinia virus immunology, Vaccinia virus pathogenicity, Viral Vaccines genetics, Cancer Vaccines therapeutic use, Communicable Diseases, Emerging drug therapy, Neoplasms drug therapy, Viral Vaccines therapeutic use
- Abstract
Recombinants based on poxviruses have been used extensively as gene delivery systems to study many biological functions of foreign genes and as vaccines against many pathogens, particularly in the veterinary field. Based on safety record, efficient expression and ability to trigger specific immune responses, two of the most promising poxvirus vectors for human use are the attenuated modified vaccinia virus Ankara (MVA) and the Copenhagen derived NYVAC strains. Because of the scientific and clinical interest in these two vectors, here we review their biological characteristics, with emphasis on virus-host cell interactions, viral immunomodulators, gene expression profiling, virus distribution in animals, and application as vaccines against different pathogens and tumors.
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- 2011
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11. T-cell immune responses against Env from CRF12_BF and subtype B HIV-1 show high clade-specificity that can be overridden by multiclade immunizations.
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Mónaco DC, Rodríguez AM, Pascutti MF, Carobene M, Falivene J, Gómez A, Maeto C, Turk G, Nájera JL, Esteban M, and Gherardi MM
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- AIDS Vaccines therapeutic use, Amino Acid Sequence, Animals, BALB 3T3 Cells, Cells, Cultured, Female, HIV Antigens genetics, HIV Antigens immunology, HIV Infections immunology, HIV Infections prevention & control, HIV-1 genetics, HeLa Cells, Humans, Mice, Mice, Inbred BALB C, Models, Biological, Molecular Sequence Data, T-Lymphocytes physiology, env Gene Products, Human Immunodeficiency Virus chemistry, HIV-1 classification, HIV-1 immunology, Immunity, Cellular immunology, Immunization methods, T-Cell Antigen Receptor Specificity immunology, T-Lymphocytes immunology, env Gene Products, Human Immunodeficiency Virus immunology
- Abstract
Background: The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors., Methodology/principal Findings: As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B., Conclusions/significance: This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate.
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- 2011
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12. Diversity in viral anti-PKR mechanisms: a remarkable case of evolutionary convergence.
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Domingo-Gil E, Toribio R, Nájera JL, Esteban M, and Ventoso I
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- Animals, Base Sequence, Cells, Cultured, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Knockout, Models, Biological, Molecular Sequence Data, Nucleic Acid Conformation, Signal Transduction genetics, Sindbis Virus genetics, Sindbis Virus metabolism, Virus Replication genetics, Viruses metabolism, Viruses pathogenicity, eIF-2 Kinase genetics, Evolution, Molecular, Genetic Variation physiology, Host-Pathogen Interactions genetics, Viruses genetics, eIF-2 Kinase antagonists & inhibitors
- Abstract
Most viruses express during infection products that prevent or neutralize the effect of the host dsRNA activated protein kinase (PKR). Translation of Sindbis virus (SINV) mRNA escapes to PKR activation and eIF2 phosphorylation in infected cells by a mechanism that requires a stem loop structure in viral 26S mRNA termed DLP to initiate translation in the absence of functional eIF2. Unlike the rest of viruses tested, we found that Alphavirus infection allowed a strong PKR activation and eIF2α phosphorylation in vitro and in infected animals so that the presence of DLP structure in mRNA was critical for translation and replication of SINV. Interestingly, infection of MEFs with some viruses that express PKR inhibitors prevented eIF2α phosphorylation after superinfection with SINV, suggesting that viral anti-PKR mechanisms could be exchangeable. Thus, translation of SINV mutant lacking the DLP structure (ΔDLP) in 26S mRNA was partially rescued in cells expressing vaccinia virus (VV) E3 protein, a known inhibitor of PKR. This case of heterotypic complementation among evolutionary distant viruses confirmed experimentally a remarkable case of convergent evolution in viral anti-PKR mechanisms. Our data reinforce the critical role of PKR in regulating virus-host interaction and reveal the versatility of viruses to find different solutions to solve the same conflict.
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- 2011
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13. A candidate HIV/AIDS vaccine (MVA-B) lacking vaccinia virus gene C6L enhances memory HIV-1-specific T-cell responses.
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García-Arriaza J, Nájera JL, Gómez CE, Tewabe N, Sorzano CO, Calandra T, Roger T, and Esteban M
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- Animals, Antibodies, Viral immunology, Chickens, Dendritic Cells immunology, Dendritic Cells virology, HIV Envelope Protein gp120 immunology, Humans, Immunity, Humoral immunology, Immunization, Interferon-beta metabolism, Macrophages immunology, Macrophages virology, Mice, Mice, Inbred C57BL, Mutation genetics, Protein Transport, Species Specificity, AIDS Vaccines immunology, Genes, Viral genetics, HIV-1 immunology, Immunologic Memory immunology, T-Lymphocytes immunology, Vaccinia virus genetics
- Abstract
The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B ΔC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B ΔC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-β and IFN-α/β-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B ΔC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4+ and CD8+ T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8+ T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8+ T-cell responses, MVA-B ΔC6L induced more Gag-Pol-Nef-specific CD8+ T-cell responses. Furthermore, MVA-B ΔC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-β-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines.
- Published
- 2011
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14. Immunogenic profiling in mice of a HIV/AIDS vaccine candidate (MVA-B) expressing four HIV-1 antigens and potentiation by specific gene deletions.
- Author
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García-Arriaza J, Nájera JL, Gómez CE, Sorzano CO, and Esteban M
- Subjects
- Animals, Antibodies, Viral immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Gene Expression, Genetic Vectors genetics, Genetic Vectors immunology, HIV Antigens immunology, HIV Envelope Protein gp120 immunology, Immunologic Memory immunology, Mice, Species Specificity, AIDS Vaccines genetics, AIDS Vaccines immunology, Gene Deletion, Genes, Viral genetics, HIV Antigens genetics, Poxviridae genetics, Poxviridae immunology
- Abstract
Background: The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function., Methodology/principal Findings: In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1beta, respectively (referred as MVA-B DeltaA41L/DeltaB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B DeltaA41L/DeltaB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4(+) and CD8(+) T cells, with the CD8(+) T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B DeltaA41L/DeltaB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+) and CD8(+) T-cell immune responses. HIV-1-specific CD4(+) T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8(+) T-cell responses, MVA-B DeltaA41L/DeltaB16R induced more GPN-specific CD8(+) T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env., Conclusions/significance: These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.
- Published
- 2010
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15. Selective induction of host genes by MVA-B, a candidate vaccine against HIV/AIDS.
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Guerra S, González JM, Climent N, Reyburn H, López-Fernández LA, Nájera JL, Gómez CE, García F, Gatell JM, Gallart T, and Esteban M
- Subjects
- AIDS Vaccines genetics, Antigen Presentation, Cells, Cultured, Cytokines biosynthesis, Endoplasmic Reticulum Chaperone BiP, Genetic Vectors, HIV Envelope Protein gp120 genetics, HIV Envelope Protein gp120 immunology, Humans, Signal Transduction genetics, Vaccinia virus genetics, gag Gene Products, Human Immunodeficiency Virus genetics, gag Gene Products, Human Immunodeficiency Virus immunology, nef Gene Products, Human Immunodeficiency Virus genetics, nef Gene Products, Human Immunodeficiency Virus immunology, pol Gene Products, Human Immunodeficiency Virus genetics, pol Gene Products, Human Immunodeficiency Virus immunology, AIDS Vaccines immunology, Dendritic Cells immunology, Gene Expression Profiling
- Abstract
The aim of this study was to define the effects on antigen-presenting cells of the expression of HIV antigens from an attenuated poxvirus vector. We have analyzed the transcriptional changes in gene expression following infection of human immature monocyte-derived dendritic cells (DC) with recombinant modified vaccinia virus Ankara (MVA) expressing the genes encoding the gp120 and Gag-Pol-Nef antigens of HIV type 1 clade B (referred to as MVA-B) versus parental MVA infection. Using microarray technology and real-time reverse transcription-PCR, we demonstrated that the HIV proteins induced the expression of cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex (MHC) genes. Levels of mRNAs for interleukin-1, beta interferon, CCR8, and SCYA20 were higher after HIV antigen production. MVA-B infection also modulated the expression of antigen processing and presentation genes: the gene for MICA was upregulated, whereas those for HLA-DRA and HSPA5 were downregulated. Indeed, the increased expression of the gene for MICA, a glycoprotein related to major histocompatibility complex class I molecules, was shown to enhance the interaction between MVA-B-infected target cells and cytotoxic lymphocytes. The expression profiles of the genes for protein kinases such as JAK1 and IRAK2 were activated after HIV antigen expression. Several genes included in the JAK-STAT and mitogen-activated protein kinase signaling pathways were regulated after HIV antigen expression. Our findings provide the first gene signatures in DC of a candidate MVA-B vaccine expressing four HIV antigens and identified the biological roles of some of the regulatory genes, like that for MICA, which will help in the design of more effective MVA-derived vaccines.
- Published
- 2010
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16. Insertion of vaccinia virus C7L host range gene into NYVAC-B genome potentiates immune responses against HIV-1 antigens.
- Author
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Nájera JL, Gómez CE, García-Arriaza J, Sorzano CO, and Esteban M
- Subjects
- Animals, Cells, Cultured, Genetic Vectors, Humans, Mice, Mice, Inbred BALB C, Vaccinia virus physiology, Virus Replication, AIDS Vaccines immunology, Genome, Viral, HIV Antigens immunology, HIV-1 immunology, Vaccinia virus genetics
- Abstract
Background: The highly attenuated vaccinia virus strain NYVAC expressing HIV-1 components has been evaluated as a vaccine candidate in preclinical and clinical trials with encouraging results. We have previously described that the presence of C7L in the NYVAC genome prevents the induction of apoptosis and renders the vector capable of replication in human and murine cell lines while maintaining an attenuated phenotype in mice., Methodology/principal Findings: In an effort to improve the immunogenicity of NYVAC, we have developed a novel poxvirus vector by inserting the VACV host-range C7L gene into the genome of NYVAC-B, a recombinant virus that expresses four HIV-1 antigens from clade B (Env, Gag, Pol and Nef) (referred as NYVAC-B-C7L). In the present study, we have compared the in vitro and in vivo behavior of NYVAC-B and NYVAC-B-C7L. In cultured cells, NYVAC-B-C7L expresses higher levels of heterologous antigen than NYVAC-B as determined by Western blot and fluorescent-activated cell sorting to score Gag expressing cells. In a DNA prime/poxvirus boost approach with BALB/c mice, both recombinants elicited robust, broad and multifunctional antigen-specific T-cell responses to the HIV-1 immunogens expressed from the vectors. However, the use of NYVAC-B-C7L as booster significantly enhanced the magnitude of the T cell responses, and induced a more balanced cellular immune response to the HIV-1 antigens in comparison to that elicited in animals boosted with NYVAC-B., Conclusions/significance: These findings demonstrate the possibility to enhance the immunogenicity of the highly attenuated NYVAC vector by the insertion of the host-range gene C7L and suggest the use of this modified vector as an improved vaccine candidate against HIV/AIDS.
- Published
- 2010
- Full Text
- View/download PDF
17. Characterization of DNA and MVA vectors expressing Nef from HIV-1 CRF12_BF revealed high immune specificity with low cross-reactivity against subtype B.
- Author
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Rodríguez AM, Turk G, Pascutti MF, Ferrer F, Nájera JL, Mónaco D, Esteban M, Salomón H, Calamante G, and Gherardi MM
- Subjects
- AIDS Vaccines genetics, Animals, Antibodies, Neutralizing blood, Cell Line, Chlorocebus aethiops, Cricetinae, Cross Reactions, Genetic Vectors, HIV Antibodies blood, HIV Infections prevention & control, HIV-1 genetics, Immunization, Secondary methods, Interferon-gamma metabolism, Leukocytes, Mononuclear immunology, Mice, Mice, Inbred BALB C, Vaccines, DNA genetics, Vaccines, Subunit genetics, Vaccines, Subunit immunology, nef Gene Products, Human Immunodeficiency Virus genetics, AIDS Vaccines immunology, HIV-1 immunology, Vaccines, DNA immunology, Vaccinia virus genetics, nef Gene Products, Human Immunodeficiency Virus immunology
- Abstract
The HIV epidemic in Argentina is characterized by the high prevalence of infections caused by subtype B and BF variants. In this study, the Nef protein was used as a tool to study the impact of HIV-1 BF variants in the design of future vaccines. DNA and MVA vectors expressing Nef of the CRF12_BF recombinant form of HIV-1 were generated and characterized. After the administration of single DNAprime/MVAboost immunization schedules in Balb/c mice we found that NefBF delivered from these vectors generated a response of high specificity with low cross-reactivity against subtype B. But, when a more potent response was induced after 3 priming DNA doses and a booster with MVA virus, cross-reactivity against NefB was detected, although of lower magnitude than the NefBF specific. These results will be pivotal for vaccines designs in our region, indicating that antigens from these viral variants must be considered for a future vaccine.
- Published
- 2009
- Full Text
- View/download PDF
18. Multimeric soluble CD40 ligand (sCD40L) efficiently enhances HIV specific cellular immune responses during DNA prime and boost with attenuated poxvirus vectors MVA and NYVAC expressing HIV antigens.
- Author
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Gómez CE, Nájera JL, Sánchez R, Jiménez V, and Esteban M
- Subjects
- Animals, CD8-Positive T-Lymphocytes immunology, Female, Genetic Vectors, HIV Antigens genetics, Immunization, Secondary, Mice, Mice, Inbred BALB C, Poxviridae genetics, AIDS Vaccines immunology, Adjuvants, Immunologic pharmacology, CD40 Ligand pharmacology, HIV Antigens immunology, HIV-1 immunology, Vaccines, DNA immunology, Vaccines, Synthetic immunology
- Abstract
The attenuated poxvirus vectors MVA and NYVAC are now in clinical trials against HIV/AIDS. Due to the vectors restricted replication capacity in human cells, approaches to enhance their immunogenicity are highly desirable. Here, we have analyzed the ability of a soluble form of hexameric CD40L (sCD40L) to stimulate specific immune responses to HIV antigens when inoculated in mice during priming with DNA and in the booster with MVA or NYVAC, expressing the vectors HIV-1 Env, Gag, Pol and Nef antigens from clade B. Our findings revealed that sCD40L in DNA/poxvirus combination enhanced the magnitude about 2-fold (DNA-B/MVA-B) and 4-fold (DNA-B/NYVAC-B), as well as the breath of the HIV antigen specific cellular immune responses. sCD40L was necessary in both prime and boost inoculations triggering a potent polarization of the Th response towards a Th1 type. In DNA-B/NYVAC-B regime the addition of sCD40L significantly enhanced the humoral immune response against HIV gp160, but not in DNA-B/MVA-B combination. These findings provided evidence for the immunostimulatory benefit of sCD40L when DNA and the poxvirus vectors MVA and NYVAC are used as immunogens.
- Published
- 2009
- Full Text
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19. The poxvirus vectors MVA and NYVAC as gene delivery systems for vaccination against infectious diseases and cancer.
- Author
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Gómez CE, Nájera JL, Krupa M, and Esteban M
- Subjects
- Animals, Communicable Diseases microbiology, Communicable Diseases parasitology, Communicable Diseases virology, Genetic Vectors genetics, Humans, Neoplasms immunology, Neoplasms prevention & control, Poxviridae immunology, Poxviridae physiology, Viral Vaccines immunology, Cancer Vaccines genetics, Cancer Vaccines immunology, Communicable Diseases immunology, Gene Transfer Techniques, Neoplasms therapy, Poxviridae genetics, Viral Vaccines genetics
- Abstract
Recombinants based on poxviruses have been used extensively as gene delivery systems to study many biological functions of foreign genes and as vaccines against many pathogens, particularly in the veterinary field. Based on safety record, efficient expression and ability to trigger specific immune responses, two of the most promising poxvirus vectors for human use are the attenuated modified vaccinia virus Ankara (MVA) and the Copenhagen derived NYVAC strains. Because of the scientific and clinical interest in these two vectors, here we review their biological characteristics, with emphasis on virus-host cell interactions, viral immunomodulators, gene expression profiling, virus distribution in animals, and application as vaccines against different pathogens and tumors.
- Published
- 2008
- Full Text
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20. Differential CD4+ versus CD8+ T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates.
- Author
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Mooij P, Balla-Jhagjhoorsingh SS, Koopman G, Beenhakker N, van Haaften P, Baak I, Nieuwenhuis IG, Kondova I, Wagner R, Wolf H, Gómez CE, Nájera JL, Jiménez V, Esteban M, and Heeney JL
- Subjects
- Animals, Enzyme-Linked Immunosorbent Assay, HIV Antigens immunology, Immunophenotyping, Macaca mulatta, Poxviridae genetics, AIDS Vaccines immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, HIV-1 immunology, Poxviridae immunology
- Abstract
Poxvirus vectors have proven to be highly effective for boosting immune responses in diverse vaccine settings. Recent reports reveal marked differences in the gene expression of human dendritic cells infected with two leading poxvirus-based human immunodeficiency virus (HIV) vaccine candidates, New York vaccinia virus (NYVAC) and modified vaccinia virus Ankara (MVA). To understand how complex genomic changes in these two vaccine vectors translate into antigen-specific systemic immune responses, we undertook a head-to-head vaccine immunogenicity and efficacy study in the pathogenic HIV type 1 (HIV-1) model of AIDS in Indian rhesus macaques. Differences in the immune responses in outbred animals were not distinguished by enzyme-linked immunospot assays, but differences were distinguished by multiparameter fluorescence-activated cell sorter analysis, revealing a difference between the number of animals with both CD4(+) and CD8(+) T-cell responses to vaccine inserts (MVA) and those that elicit a dominant CD4(+) T-cell response (NYVAC). Remarkably, vector-induced differences in CD4(+)/CD8(+) T-cell immune responses persisted for more than a year after challenge and even accompanied antigenic modulation throughout the control of chronic infection. Importantly, strong preexposure HIV-1/simian immunodeficiency virus-specific CD4(+) T-cell responses did not prove deleterious with respect to accelerated disease progression. In contrast, in this setting, animals with strong vaccine-induced polyfunctional CD4(+) T-cell responses showed efficacies similar to those with stronger CD8(+) T-cell responses.
- Published
- 2008
- Full Text
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21. Expression of the E3L gene of vaccinia virus in transgenic mice decreases host resistance to vaccinia virus and Leishmania major infections.
- Author
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Domingo-Gil E, Pérez-Jiménez E, Ventoso I, Nájera JL, and Esteban M
- Subjects
- Animals, Body Weight, Cell Line, Female, Immunity, Innate immunology, Leishmania major immunology, Liver virology, Lymph Nodes immunology, Lymphocyte Subsets immunology, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Transgenic, Ovary virology, Parasitemia, RNA-Binding Proteins genetics, Spleen immunology, Spleen virology, T-Lymphocytes immunology, Tail pathology, Vaccinia virus immunology, Viral Proteins genetics, Immunity, Innate genetics, Leishmaniasis, Cutaneous immunology, RNA-Binding Proteins biosynthesis, Vaccinia immunology, Viral Proteins biosynthesis
- Abstract
The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2'-5'-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.
- Published
- 2008
- Full Text
- View/download PDF
22. Virus distribution of the attenuated MVA and NYVAC poxvirus strains in mice.
- Author
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Gómez CE, Nájera JL, Domingo-Gil E, Ochoa-Callejero L, González-Aseguinolaza G, and Esteban M
- Subjects
- Animals, Genes, Reporter, Kinetics, Luciferases genetics, Mice, Poxviridae drug effects, Vaccines, Attenuated pharmacology, Poxviridae immunology, Viral Vaccines pharmacology
- Abstract
Recombinant vaccinia viruses based on the attenuated NYVAC and MVA strains are promising vaccine candidates against a broad spectrum of diseases. Whilst these vectors are safe and immunogenic in animals and humans, little is known about their comparative behaviour in vivo. In this investigation, a head-to-head analysis was carried out of virus dissemination in mice inoculated by the mucosal or systemic route with replication-competent (WRluc) and attenuated recombinant (MVAluc and NYVACluc) viruses expressing the luciferase gene. Bioluminescence imaging showed that, in contrast to WRluc, the attenuated recombinants expressed the reporter gene transiently, with MVAluc expression limited to the first 24 h and NYVACluc giving a longer signal, up to 72 h post-infection, for most of the routes assayed. Moreover, luciferase levels in MVAluc-infected tissues peaked earlier than those in tissues infected by NYVACluc. These findings may be of immunological relevance when these vectors are used as recombinant vaccines.
- Published
- 2007
- Full Text
- View/download PDF
23. Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.
- Author
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Guerra S, Nájera JL, González JM, López-Fernández LA, Climent N, Gatell JM, Gallart T, and Esteban M
- Subjects
- Apoptosis genetics, Cells, Cultured, Cytokines genetics, Gene Expression Regulation, Genetic Vectors genetics, HeLa Cells, Humans, Monocytes immunology, Oligonucleotide Array Sequence Analysis, Vaccines, Attenuated immunology, Vaccinia virus genetics, Dendritic Cells immunology, Dendritic Cells virology, Gene Expression Profiling, Genetic Vectors immunology, Vaccinia virus immunology
- Abstract
Although recombinants based on the attenuated poxvirus vectors MVA and NYVAC are currently in clinical trials, the nature of the genes triggered by these vectors in antigen-presenting cells is poorly characterized. Using microarray technology and various analysis conditions, we compared specific changes in gene expression profiling following MVA and NYVAC infection of immature human monocyte-derived dendritic cells (MDDC). Microarray analysis was performed at 6 h postinfection, since these viruses induced extensive cytopathic effects, rRNA breakdown, and apoptosis at late times postinfection. MVA- and NYVAC-infected MDDC shared upregulation of 195 genes compared to uninfected cells: MVA specifically upregulated 359 genes, and NYVAC upregulated 165 genes. Microarray comparison of NYVAC and MVA infection revealed 544 genes with distinct expression patterns after poxvirus infection and 283 genes specifically upregulated after MVA infection. Both vectors upregulated genes for cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex genes. mRNA levels for interleukin 12beta (IL-12beta), beta interferon, and tumor necrosis factor alpha were higher after MVA infection than after NYVAC infection. The expression profiles of transcription factors such as NF-kappaB/Rel and STAT were regulated similarly by both viruses; in contrast, OASL, MDA5, and IRIG-I expression increased only during MVA infection. Type I interferon, IL-6, and Toll-like receptor pathways were specifically induced after MVA infection. Following MVA or NYVAC infection in MDDC, we found similarities as well as differences between these virus strains in the expression of cellular genes with immunological function, which should have an impact when these vectors are used as recombinant vaccines.
- Published
- 2007
- Full Text
- View/download PDF
24. Head-to-head comparison on the immunogenicity of two HIV/AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1BX08 gp120 and HIV-1(IIIB) Gag-Pol-Nef proteins of clade B.
- Author
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Gómez CE, Nájera JL, Jiménez EP, Jiménez V, Wagner R, Graf M, Frachette MJ, Liljeström P, Pantaleo G, and Esteban M
- Subjects
- AIDS Vaccines genetics, Animals, Antigens, Viral biosynthesis, Antigens, Viral genetics, Apoptosis immunology, Base Sequence, Chick Embryo, Fusion Proteins, gag-pol biosynthesis, Fusion Proteins, gag-pol genetics, Fusion Proteins, gag-pol immunology, Gene Products, nef biosynthesis, Gene Products, nef genetics, Gene Products, nef immunology, Genomic Instability, HIV Envelope Protein gp120 biosynthesis, HIV Envelope Protein gp120 genetics, HLA-A2 Antigen immunology, HeLa Cells, Humans, Mice, Mice, Inbred BALB C, Mice, Transgenic, Molecular Sequence Data, Polymerase Chain Reaction methods, Poxviridae genetics, Poxviridae immunology, Vaccines, Attenuated genetics, Vaccines, Attenuated immunology, Viral Vaccines genetics, AIDS Vaccines immunology, Antigens, Viral immunology, HIV Envelope Protein gp120 immunology, Viral Vaccines immunology
- Abstract
In this investigation we have generated and defined the immunogenicity of two novel HIV/AIDS vaccine candidates based on the highly attenuated vaccinia virus strains, MVA and NYVAC, efficiently expressing in the same locus (TK) and under the same viral promoter the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef antigens of clade B (referred as MVA-B and NYVAC-B). In infected human HeLa cells, gp120 is released from cells and GPN is produced as a polyprotein; NYVAC-B induces severe apoptosis but not MVA-B. The two poxvirus vectors showed genetic stability of the inserts. In BALB/c and in transgenic HHD mice for human HLA-A2 class I, both vectors are efficient immunogens and induced broad cellular immune responses against peptides represented in the four HIV-1 antigens. Some differences were observed in the magnitude and breadth of the immune response in the mouse models. In DNA prime/poxvirus boost protocols, the strongest immune response, as measured by fresh IFN-gamma and IL-2 ELISPOT, was obtained in BALB/c mice boosted with NYVAC-B, while in HHD mice there were no differences between the poxvirus vectors. When the prime/boost was performed with homologous or with combination of poxvirus vectors, the protocols MVA-B/MVA-B and NYVAC-B/NYVAC-B, or the combination NYVAC-B/MVA-B gave the most consistent broader immune response in both mouse models, although the magnitude of the overall response was higher for the DNA-B/poxvirus-B regime. All of the immunization protocols induced some humoral response against the gp160 protein from HIV-1 clone LAV. Our findings indicate that MVA-B and NYVAC-B meet the criteria to be potentially useful vaccine candidates against HIV/AIDS.
- Published
- 2007
- Full Text
- View/download PDF
25. Generation and immunogenicity of novel HIV/AIDS vaccine candidates targeting HIV-1 Env/Gag-Pol-Nef antigens of clade C.
- Author
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Gómez CE, Nájera JL, Jiménez V, Bieler K, Wild J, Kostic L, Heidari S, Chen M, Frachette MJ, Pantaleo G, Wolf H, Liljeström P, Wagner R, and Esteban M
- Subjects
- AIDS Vaccines genetics, Animals, Base Sequence, Codon genetics, Gene Products, gag genetics, Gene Products, gag immunology, Gene Products, nef genetics, Gene Products, nef immunology, Gene Products, pol genetics, Gene Products, pol immunology, Genetic Vectors, HIV Antigens genetics, HIV Envelope Protein gp120 genetics, HIV Envelope Protein gp120 immunology, HIV-1 genetics, Humans, Immunization, Secondary, Interferon-gamma biosynthesis, Interleukin-2 biosynthesis, Mice, Mice, Inbred BALB C, Mice, Transgenic, Models, Animal, Molecular Sequence Data, Semliki forest virus, Spleen immunology, T-Lymphocytes immunology, Vaccines, DNA genetics, Vaccines, DNA immunology, Vaccinia virus, Viral Vaccines, nef Gene Products, Human Immunodeficiency Virus, AIDS Vaccines immunology, HIV Antigens immunology, HIV-1 immunology
- Abstract
Recombinants based on the attenuated vaccinia virus strains MVA and NYVAC are considered candidate vectors against different human diseases. In this study we have generated and characterized in BALB/c and in transgenic HHD mice the immunogenicity of two attenuated poxvirus vectors expressing in a single locus (TK) the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef (GPN) polyprotein of clade C (referred as MVA-C and NYVAC-C). In HHD mice primed with either MVA-C or NYVAC-C, or primed with DNA-C and boosted with the poxvirus vectors, the splenic T cell responses against clade C peptides spanning gp120/GPN was broad and mainly directed against Gag-1, Env-1 and Env-2 peptide pools. In BALB/c mice immunized with the homologous or the heterologous combination of poxvirus vectors or with Semliki forest virus (SFV) vectors expressing gp120/GPN, the immune response was also broad but the most immunogenic peptides were Env-1, GPN-1 and GPN-2. Differences in the magnitude of the cellular immune responses were observed between the poxvirus vectors depending on the protocol used. The specific cellular immune response triggered by the poxvirus vectors was Th1 type. The cellular response against the vectors was higher for NYVAC than for MVA in both HHD and BALB/c mice, but differences in viral antigen recognition between the vectors was observed in sera from the poxvirus-immunized animals. These results demonstrate the immunogenic potential of MVA-C and NYVAC-C as novel vaccine candidates against clade C of HIV-1.
- Published
- 2007
- Full Text
- View/download PDF
26. Cellular and biochemical differences between two attenuated poxvirus vaccine candidates (MVA and NYVAC) and role of the C7L gene.
- Author
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Nájera JL, Gómez CE, Domingo-Gil E, Gherardi MM, and Esteban M
- Subjects
- Animals, Apoptosis, HeLa Cells, Humans, Mice, Poxviridae Infections, Vaccines, Attenuated, Virus Replication, Genes, Viral physiology, Poxviridae pathogenicity, Viral Vaccines
- Abstract
The poxvirus strains NYVAC and MVA are two candidate vectors for the development of vaccines against a broad spectrum of diseases. Although these attenuated virus strains have proven to be safe in animals and humans, little is known about their comparative behavior in vitro. In contrast with MVA, NYVAC infection triggers greater cytopathic effect in a range of permissive and nonpermissive cell lines. The yields of NYVAC cell-associated virus in permissive cells (BHK-21) were slightly reduced compared with those of MVA infection. During the course of infection in HeLa cells, there is a translational block induced by NYVAC late in infection, which correlated with a marked increase in phosphorylation levels of the initiation factor eIF-2alpha. In contrast to MVA, the synthesis of certain late viral proteins was only blocked in NYVAC-infected HeLa cells. Electron microscopy (EM) analysis revealed that morphogenesis of NYVAC in HeLa cells was blocked at the stage of formation of immature viral forms. Phase-contrast microscopy, EM, flow cytometry, and rRNA analyses demonstrated that contrary to MVA, NYVAC infection induces potent apoptosis, a phenomenon dependent on activation of caspases and RNase L. Apoptosis induced by NYVAC was prevented when the virus gene C7L was placed back into the NYVAC genome, recovering the ability of NYVAC to replicate in HeLa cells and maintaining the attenuated phenotype in mice. Overall, our findings demonstrate distinct behavior between NYVAC and MVA strains in cultured cells, as well as a new role for the C7L viral gene as an inhibitor of apoptosis in NYVAC infection.
- Published
- 2006
- Full Text
- View/download PDF
27. Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.
- Author
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Guerra S, López-Fernández LA, Pascual-Montano A, Nájera JL, Zaballos A, and Esteban M
- Subjects
- Activating Transcription Factors genetics, Activating Transcription Factors metabolism, Blood Proteins genetics, Blood Proteins metabolism, Caspase 9, Caspases genetics, Caspases metabolism, Gene Expression Profiling, HeLa Cells metabolism, Humans, NF-kappa B genetics, Protein Array Analysis, Up-Regulation, Apoptosis genetics, NF-kappa B metabolism, Vaccinia virus physiology
- Abstract
NYVAC has been engineered as a safe, attenuated vaccinia virus (VV) vector for use in vaccination against a broad spectrum of pathogens and tumors. Due to the interest in NYVAC-based vectors as vaccines and current phase I/II clinical trials with this vector, there is a need to analyze the human host response to NYVAC infection. Using high-density cDNA microarrays, we found 368 differentially regulated genes after NYVAC infection of HeLa cells. Clustering of the regulated genes identified six discrete gene clusters with altered expression patterns. Clusters 1 to 3 represented 47.5% of the regulated genes, with three patterns of gene activation kinetics, whereas clusters 4 to 6 showed distinct repression kinetics. Quantitative real-time reverse transcription-PCR analysis of selected genes validated the array data. Upregulated transcripts correlated with genes implicated in immune responses, including those encoding interleukin-1 receptor 2 (IL-1R2), IL-6, ISG-15, CD-80, and TNFSF7. NYVAC upregulated several intermediates of apoptotic cascades, including caspase-9, correlating with its ability to induce apoptosis. NYVAC infection also stimulated the expression of NF-kappaB1 and NF-kappaB2 as well as that of NF-kappaB target genes. Expression of the VV host range K1L gene during NYVAC infection prevented NF-kappaB activation, but not the induction of apoptosis. This study is the first overall analysis of the transcriptional response of human cells to NYVAC infection and provides a framework for future functional studies to evaluate this vector and its derivatives as human vaccines.
- Published
- 2006
- Full Text
- View/download PDF
28. Induction of HIV immunity in the genital tract after intranasal delivery of a MVA vector: enhanced immunogenicity after DNA prime-modified vaccinia virus Ankara boost immunization schedule.
- Author
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Gherardi MM, Pérez-Jiménez E, Nájera JL, and Esteban M
- Subjects
- AIDS Vaccines genetics, AIDS Vaccines immunology, Adjuvants, Immunologic genetics, Administration, Intranasal, Administration, Intravaginal, Animals, Cholera Toxin administration & dosage, Cholera Toxin immunology, Female, Gene Products, env biosynthesis, Gene Products, env immunology, Genetic Vectors, HIV Antibodies biosynthesis, HIV-1 genetics, Immunity, Cellular, Immunity, Mucosal genetics, Interferon-gamma metabolism, Lymph Nodes immunology, Lymph Nodes metabolism, Lymph Nodes virology, Mice, Mice, Inbred BALB C, Rectum immunology, Rectum pathology, Rectum virology, Urogenital System virology, Vaccines, DNA genetics, Vaccines, DNA immunology, Vaccines, Synthetic administration & dosage, Vaccines, Synthetic genetics, Vaccines, Synthetic immunology, Vaccinia virus immunology, Vaccinia virus physiology, Virus Replication immunology, AIDS Vaccines administration & dosage, Adjuvants, Immunologic administration & dosage, HIV-1 immunology, Immunization Schedule, Immunization, Secondary methods, Urogenital System immunology, Vaccines, DNA administration & dosage, Vaccinia virus genetics
- Abstract
Vaccines intended to prevent mucosal transmission of HIV should be able to induce multiple immune effectors in the host including Abs and cell-mediated immune responses at mucosal sites. The aim of this study was to characterize and to enhance the immunogenicity of a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 Env IIIB Ag (MVAenv) inoculated in BALB/c mice by mucosal routes. Intravaginal inoculation of MVAenv was not immunogenic, whereas intranasally it induced a significant immune response to the HIV Ag. Intranasal codelivery of MVAenv plus cholera toxin (CT) significantly enhanced the cellular and humoral immune response against Env in the spleen and genitorectal draining lymph nodes, respectively. Heterologous DNAenv prime-MVAenv boost by intranasal immunization, together with CT, produced a cellular immune response in the spleen 10-fold superior to that in the absence of CT. A key finding of these studies was that both MVAenv/MVAenv and DNAenv/MVAenv schemes, plus CT, induced a specific mucosal CD8(+) T cell response in genital tissue and draining lymph nodes. In addition, both immunizations also generated systemic Abs, and more importantly, mucosal IgA and IgG Abs in vaginal washings. Specific secretion of beta-chemokines was also generated by both immunizations, with a stronger response in mice immunized by the DNA-CT/MVA-CT regimen. Our findings are of relevance in the area of vaccine development and support the optimization of protocols of immunization based on MVA as vaccine vectors to induce mucosal immune responses against HIV.
- Published
- 2004
- Full Text
- View/download PDF
29. Prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus Env protein systemically and in the genitorectal draining lymph nodes.
- Author
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Gherardi MM, Nájera JL, Pérez-Jiménez E, Guerra S, García-Sastre A, and Esteban M
- Subjects
- 3T3 Cells, AIDS Vaccines immunology, Animals, CD8-Positive T-Lymphocytes immunology, Cell Line, Gene Products, env genetics, Genetic Vectors immunology, Genitalia immunology, HIV Antibodies blood, HIV Envelope Protein gp120 genetics, HIV Envelope Protein gp120 immunology, HIV Infections prevention & control, Humans, Immunization Schedule, Immunization, Secondary, Lymph Nodes immunology, Lymphocyte Activation, Mice, Mice, Inbred BALB C, Orthomyxoviridae genetics, Peptide Fragments genetics, Peptide Fragments immunology, Recombination, Genetic, Rectum immunology, Vaccinia virus genetics, AIDS Vaccines administration & dosage, Gene Products, env immunology, Genetic Vectors administration & dosage, Orthomyxoviridae immunology, Vaccinia virus immunology
- Abstract
Vaccines that elicit systemic and mucosal immune responses should be the choice to control human immunodeficiency virus (HIV) infections. We have previously shown that prime-boost immunizations with influenza virus Env and vaccinia virus (VV) WR Env recombinants induced an enhanced systemic CD8(+) T-cell response against HIV-1 Env antigen. In this report, we analyzed in BALB/c mice after priming with influenza virus Env the ability of two VV recombinants expressing HIV-1 Env B (VV WR Env and the highly attenuated modified VV Ankara [MVA] Env) to boost cellular immune responses in the spleen and in the lymph nodes draining the genital and rectal tracts. Groups of mice were primed by the intranasal route with 10(4) PFU of influenza virus Env and boosted 14 days later by the intraperitoneal or intranasal route with 10(7) PFU of MVA Env or VV WR Env, while the control group received two immunizations with influenza virus Env. We found that the combined immunization (Flu/VV) increased more than 60 times the number of gamma interferon-specific CD8(+) T cells compared to the Flu/Flu scheme. Significantly, boosting with MVA Env by the intraperitoneal route induced a response 1.25 or 2.5 times (spleen or genital lymph nodes) higher with respect to that found after the boost with VV WR Env. Mice with an enhanced CD8(+) T-cell response also had an increased Th1/Th2 ratio, evaluated by the cytokine pattern secreted following in vitro restimulation with gp160 protein and by the specific immunoglobulin G2a (IgG2a)/IgG1 ratio in serum. By the intranasal route recombinant WR Env booster gave a more efficient immune response (10 and 1.3 times in spleen and genital lymph nodes, respectively) than recombinant MVA Env. However, the scheme influenza virus Env/MVA Env increased four times the response in the spleen, giving a low but significant response in the genital lymph nodes compared with a single intranasal immunization with MVA Env. These results demonstrate that the combination Flu/MVA in prime-booster immunization regimens is an effective vaccination approach to generate cellular immune responses to HIV antigens at sites critical for protective responses.
- Published
- 2003
- Full Text
- View/download PDF
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