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Find video protocols related to scientific articles indexed in Pubmed.
Virological and immunological characterization of novel NYVAC-based HIV/AIDS vaccine candidates expressing clade C trimeric soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs.
J. Virol.
PUBLISHED: 10-31-2014
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The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation and vaccine regimen. In this study, we have generated two novel NYVAC vectors expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef) and defined their virological and immunological characteristics in cultured cells and in mice. Insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells, Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights in virus-host cell interactions of NYVAC vectors expressing HIV antigens, with activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors.
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Clinical applications of attenuated MVA poxvirus strain.
Expert Rev Vaccines
PUBLISHED: 10-30-2013
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The highly attenuated poxvirus strain modified vaccinia virus Ankara (MVA) has reached maturity as a vector delivery system and as a vaccine candidate against a broad spectrum of diseases. This has been largely recognized from research on virus-host cell interactions and immunological studies in pre-clinical and clinical trials. This review addresses the studies of MVA vectors used in phase I/II clinical trials, with the aim to provide the main findings obtained on their behavior when tested against relevant human diseases and cancer and also highlights the strategies currently implemented to improve the MVA immunogenicity. The authors assess that MVA vectors are progressing as strong vaccine candidates either alone or when administered in combination with other vectors.
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New vaccinia virus promoter as a potential candidate for future vaccines.
J. Gen. Virol.
PUBLISHED: 09-28-2013
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Here we describe the design and strength of a new synthetic late-early optimized (LEO) vaccinia virus (VACV) promoter used as a transcriptional regulator of GFP expression during modified vaccinia Ankara infection. In contrast to the described synthetic VACV promoter (pS), LEO induced significantly higher levels of GFP expression in vitro within the first hour after infection, which correlated with an enhancement in the GFP-specific CD8 T-cell response detected in vivo, demonstrating its potential use in future vaccines.
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Attenuated and replication-competent vaccinia virus strains M65 and M101 with distinct biology and immunogenicity as potential vaccine candidates against pathogens.
J. Virol.
PUBLISHED: 04-17-2013
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Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4(+) and CD8(+) T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4(+) whereas DNA-LACK/M101-LACK preferentially induced CD8(+) T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.
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Deletion of the vaccinia virus gene A46R, encoding for an inhibitor of TLR signalling, is an effective approach to enhance the immunogenicity in mice of the HIV/AIDS vaccine candidate NYVAC-C.
PLoS ONE
PUBLISHED: 01-01-2013
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Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-?A46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.
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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.
J. Virol.
PUBLISHED: 08-24-2011
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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-?) 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.
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Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+) T cell responses in HIV-1-infected individuals.
PLoS ONE
PUBLISHED: 04-06-2011
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Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-?, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1?, MIP-1?, RANTES, IP-10, MIG, and IFN-?). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-?, IL-2, TNF-?, MIP-1?, MIP-1?, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.
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Insertion of vaccinia virus C7L host range gene into NYVAC-B genome potentiates immune responses against HIV-1 antigens.
PLoS ONE
PUBLISHED: 03-18-2010
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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.
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Deletion of the viral anti-apoptotic gene F1L in the HIV/AIDS vaccine candidate MVA-C enhances immune responses against HIV-1 antigens.
PLoS ONE
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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-?F1L). 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-?F1L 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.
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Poxvirus vectors as HIV/AIDS vaccines in humans.
Hum Vaccin Immunother
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The RV144 phase III clinical trial with the combination of the poxvirus vector ALVAC and the HIV gp120 protein has taught us that a vaccine against HIV/AIDS is possible but further improvements are still needed. Although the HIV protective effect of RV144 was modest (31.2%), these encouraging results reinforce the use of poxvirus vectors as HIV/AIDS vaccine candidates. In this review we focus on the prophylactic clinical studies thus far performed with the more widely studied poxvirus vectors, ALVAC, MVA, NYVAC and fowlpox expressing HIV antigens. We describe the characteristics of each vector administered either alone or in combination with other vectors, with emphasis on the immune parameters evaluated in healthy volunteers, percentage of responders and triggering of humoral and T cell responses. Some of these immunogens induced broad, polyfunctional and long-lasting CD4(+) and CD8(+) T cell responses to HIV-1 antigens in most volunteers, with preference for effector memory T cells, and neutralizing antibodies, immune parameters that might be relevant in protection. Finally, we consider improvements in immunogenicity of the poxvirus vectors by the selective deletion of viral immunomodulatory genes and insertion of host range genes in the poxvirus genome. Overall, the poxvirus vectors have proven to be excellent HIV/AIDS vaccine candidates, with distinct behavior among them, and the future implementation will be dictated by their optimized immune profile in clinical trials.
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High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection.
PLoS ONE
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Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.
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Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.
PLoS ONE
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Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.
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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.
J. Virol.
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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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