scholarly journals Novel Modified Vaccinia Virus Ankara Vector Expressing Anti-apoptotic GeneB13RDelays Apoptosis and Enhances Humoral Responses

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Lynette S. Chea ◽  
Linda S. Wyatt ◽  
Sailaja Gangadhara ◽  
Bernard Moss ◽  
Rama R. Amara
Keyword(s):  
Vaccinia Virus ◽  
Vaccine Development ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus ◽  
Antibody Titers ◽  
Infected Cells ◽  
Induced Apoptosis ◽  
Humoral Responses ◽  
Antigen Presenting

ABSTRACTModified vaccinia virus Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases, including human immunodeficiency virus (HIV). MVA is highly immunogenic and elicits strong cellular and humoral responses in preclinical models and humans. However, there is potential to further enhance the immunogenicity of MVA, as MVA-infected cells undergo rapid apoptosis, leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13Rby replacing the fragmented181R/182Rgenes of MVA with a functional anti-apoptotic gene,B13R, and confirmed its anti-apoptotic function against chemically induced apoptosisin vitro. In addition, MVA-B13Rshowed a significant delay in induction of apoptosis in muscle cells derived from mice and humans, as well as in plasmacytoid dendritic cells (pDCs) and CD141+DCs from rhesus macaques, compared to the induction of apoptosis in MVA-infected cells. MVA-B13Rexpressing simian immunodeficiency virus (SIV) Gag and Pol and HIV envelope (SHIV) (MVA-B13R/SHIV) produced higher levels of envelope in the supernatants than MVA/SHIV-infected DF-1 cellsin vitro. Immunization of BALB/c mice showed induction of higher levels of envelope-specific antibody-secreting cells and memory B cells, higher IgG antibody titers, and better persistence of antibody titers with MVA-B13R/SHIV than with MVA/SHIV. Gene set enrichment analysis of draining lymph node cells from day 1 after immunization showed negative enrichment for interferon responses in MVA-B13R/SHIV-immunized mice compared to the responses in MVA/SHIV-immunized mice. Taken together, these results demonstrate that restoringB13Rfunctionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cellsin vitroand augments vaccine-induced humoral immunity in mice.IMPORTANCEMVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector, MVA-B13R, by replacing the fragmented anti-apoptotic genes181R/182Rwith a functional version derived from vaccinia virus,B13R. Our results show that MVA-B13Rsignificantly delays apoptosis in antigen-presenting cells and muscle cellsin vitroand augments vaccine-induced humoral immunity in mice, leading to the development of a novel vector for vaccine development against infectious diseases and cancer.

scholarly journals A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans

2004 ◽  
Vol 85 (4) ◽  
pp. 911-919 ◽  
Author(s):  
Matilu Mwau ◽  
Inese Cebere ◽  
Julian Sutton ◽  
Priscilla Chikoti ◽  
Nicola Winstone ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Clinical Trials ◽  
Vaccinia Virus ◽  
Mononuclear Cells ◽  
Vaccine Development ◽  
Cell Mediated Immunity ◽  
Phase I Clinical Trials ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus ◽  
Hiv 1

The immunogenicities of candidate DNA- and modified vaccinia virus Ankara (MVA)-vectored human immunodeficiency virus (HIV) vaccines were evaluated on their own and in a prime–boost regimen in phase I clinical trials in healthy uninfected individuals in the United Kingdom. Given the current lack of approaches capable of inducing broad HIV-neutralizing antibodies, the pTHr.HIVA DNA and MVA.HIVA vaccines focus solely on the induction of cell-mediated immunity. The vaccines expressed a common immunogen, HIVA, which consists of consensus HIV-1 clade A Gag p24/p17 proteins fused to a string of clade A-derived epitopes recognized by cytotoxic T lymphocytes (CTLs). Volunteers' fresh peripheral blood mononuclear cells were tested for HIV-specific responses in a validated gamma interferon enzyme-linked immunospot (ELISPOT) assay using four overlapping peptide pools across the Gag domain and three pools of known CTL epitopes present in all of the HIVA protein. Both the DNA and the MVA vaccines alone and in a DNA prime–MVA boost combination were safe and induced HIV-specific responses in 14 out of 18, seven out of eight and eight out of nine volunteers, respectively. These results are very encouraging and justify further vaccine development.

scholarly journals Direct Comparison of Antigen Production and Induction of Apoptosis by Canarypox Virus- and Modified Vaccinia Virus Ankara-Human Immunodeficiency Virus Vaccine Vectors

2007 ◽  
Vol 81 (13) ◽  
pp. 7022-7033 ◽  
Author(s):  
Xiugen Zhang ◽  
Farah Cassis-Ghavami ◽  
Mike Eller ◽  
Jeff Currier ◽  
Bonnie M. Slike ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Vaccinia Virus ◽  
Mammalian Cells ◽  
Apoptosis Induction ◽  
Antigen Production ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus ◽  
Host Cell Interactions ◽  
Infected Cells ◽  
Canarypox Virus

ABSTRACT Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles.

scholarly journals Effective Induction of Simian Immunodeficiency Virus-Specific Cytotoxic T Lymphocytes in Macaques by Using a Multiepitope Gene and DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccination Regimen

1999 ◽  
Vol 73 (9) ◽  
pp. 7524-7532 ◽  
Author(s):  
Tomas Hanke ◽  
Rachel V. Samuel ◽  
Tom J. Blanchard ◽  
Veronica C. Neumann ◽  
Todd M. Allen ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Simian Immunodeficiency Virus ◽  
Rhesus Macaques ◽  
Peripheral Blood Lymphocytes ◽  
Vaccination Regimen ◽  
Boost Regimen ◽  
Boost Vaccination ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus

ABSTRACT DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8+ lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed.

scholarly journals Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Arwen F. Altenburg ◽  
Carolien E. van de Sandt ◽  
Bobby W. S. Li ◽  
Ronan J. MacLoughlin ◽  
Ron A. M. Fouchier ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Ex Vivo ◽  
Antigen Presenting Cells ◽  
Modified Vaccinia Virus Ankara ◽  
Antigen Presenting

scholarly journals Induction of simian immunodeficiency virus (SIV)-specific CTL in rhesus macaques by vaccination with modified vaccinia virus Ankara expressing SIV transgenes: influence of pre-existing anti-vector immunity

2001 ◽  
Vol 82 (9) ◽  
pp. 2215-2223 ◽  
Author(s):  
Sally Sharpe ◽  
Natasha Polyanskaya ◽  
Mike Dennis ◽  
Gerd Sutter ◽  
Tomáš Hanke ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Simian Immunodeficiency Virus ◽  
Vaccine Development ◽  
Cytotoxic T Lymphocyte ◽  
Rhesus Macaques ◽  
Lymphoid Tissues ◽  
Specific Response ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus ◽  
Definition Of

A major aim in AIDS vaccine development is the definition of strategies to stimulate strong and durable cytotoxic T lymphocyte (CTL) responses. Here we report that simian immunodeficiency virus (SIV)-specific CTL developed in 4/4 macaques following a single intramuscular injection of modified vaccinia virus Ankara (MVA) constructs expressing both structural and regulatory/accessory genes of SIV. In two animals Nef-specific responses persisted, but other responses diminished and new responses were not revealed, following further vaccination. Vaccination of another two macaques, expressing Mamu A*01 MHC class I, with MVA constructs containing nef and gag–pol under the control of the moderate strength natural vaccinia virus early/late promoter P7.5, again induced an early Nef-specific response, whereas responses to Gag remained undetectable. Anti-vector immunity induced by this immunization was shown to prevent the efficient stimulation of CTL directed to the cognate Gag epitope, p11C C-M, following vaccination with another MVA construct expressing SIV Gag–Pol under a strong synthetic vaccinia virus-specific promoter. In contrast, vaccination of a previously unexposed animal resulted in a SIV-specific CTL response widely disseminated in lymphoid tissues including lymph nodes associated with the rectal and genital routes of SIV entry. Thus, despite the highly attenuated nature of MVA, repeated immunization may elicit sufficient anti-vector immunity to limit the effectiveness of later vaccination.

scholarly journals Critical Role for Env as well as Gag-Pol in Control of a Simian-Human Immunodeficiency Virus 89.6P Challenge by a DNA Prime/Recombinant Modified Vaccinia Virus Ankara Vaccine

2002 ◽  
Vol 76 (12) ◽  
pp. 6138-6146 ◽  
Author(s):  
Rama Rao Amara ◽  
James M. Smith ◽  
Silvija I. Staprans ◽  
David C. Montefiori ◽  
Francois Villinger ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Immune Responses ◽  
Vaccinia Virus ◽  
Critical Role ◽  
Viral Control ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus ◽  
Aids Vaccines ◽  
Induced Apoptosis ◽  
Cellular Immune

ABSTRACT Cellular immune responses against epitopes in conserved Gag and Pol sequences of human immunodeficiency virus type 1 have become popular targets for candidate AIDS vaccines. Recently, we used a simian-human immunodeficiency virus model (SHIV 89.6P) with macaques to demonstrate the control of a pathogenic mucosal challenge by priming with Gag-Pol-Env-expressing DNA and boosting with Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (rMVA). Here we tested Gag-Pol DNA priming and Gag-Pol rMVA boosting to evaluate the contribution of anti-Env immune responses to viral control. The Gag-Pol vaccine raised frequencies of Gag-specific T cells similar to those raised by the Gag-Pol-Env vaccine. Following challenge, these rapidly expanded to counter the challenge infection. Despite this, the control of the SHIV 89.6P challenge was delayed and inconsistent in the Gag-Pol-vaccinated group and all of the animals underwent severe and, in most cases, sustained loss of CD4+ cells. Interestingly, most of the CD4+ cells that were lost in the Gag-Pol-vaccinated group were uninfected cells. We suggest that the rapid appearance of binding antibody for Env in Gag-Pol-Env-vaccinated animals helped protect uninfected CD4+ cells from Env-induced apoptosis. Our results highlight the importance of immune responses to Env, as well as to Gag-Pol, in the control of immunodeficiency virus challenges and the protection of CD4+ cells.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

scholarly journals Macrophage-dependent Apoptosis of CD4+ T Lymphocytes from HIV-infected Individuals Is Mediated by FasL and Tumor Necrosis Factor

1997 ◽  
Vol 185 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Andrew D. Badley ◽  
David Dockrell ◽  
Margaret Simpson ◽  
Ron Schut ◽  
David H. Lynch ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Necrosis Factor ◽  
T Lymphocytes ◽  
Cd4 T Cells ◽  
Tumor Necrosis ◽  
Human Macrophages ◽  
Infected Cells ◽  
Induced Apoptosis ◽  
Antigen Presenting ◽  
Necrosis Factor

Apoptosis of bystander uninfected CD4+ T lymphocytes by neighboring HIV-infected cells is observed in cell culture and in lymphoid tissue of HIV-infected individuals. This study addresses whether antigen-presenting cells such as human macrophages mediate apoptosis of CD4+ T cells from HIV-infected individuals. Uninfected human macrophages, and to a larger degree, HIV-infected macrophages mediate apoptosis of T cells from HIV-infected, but not from uninfected control individuals. This macrophage-dependent killing targets CD4+, but not CD8+ T lymphocytes from HIV-infected individuals, and direct contact between macrophages and lymphocytes is required. Additional analyses indicated that the apoptosis-inducing ligands, FasL and tumor necrosis factor (TNF), mediate this macrophage-induced apoptosis of CD4+ T cells. These results support a role for macrophage-associated FasL and TNF in the selective depletion of CD4+ T cells in HIV-infected individuals.

scholarly journals The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

2002 ◽  
Vol 76 (3) ◽  
pp. 1015-1024 ◽  
Author(s):  
Barbara Müller ◽  
Tilo Patschinsky ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Metabolic Labeling ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
A Minor ◽  
Hiv 1

ABSTRACT The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho- 32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.

scholarly journals Modified Vaccinia Virus Ankara Triggers Chemotaxis of Monocytes and Early Respiratory Immigration of Leukocytes by Induction of CCL2 Expression

2009 ◽  
Vol 83 (6) ◽  
pp. 2540-2552 ◽  
Author(s):  
Michael H. Lehmann ◽  
Wolfgang Kastenmuller ◽  
Judith D. Kandemir ◽  
Florian Brandt ◽  
Yasemin Suezer ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Viral Vector ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Human Monocytic Cell Line ◽  
Modified Vaccinia Virus Ankara ◽  
Ccl2 Expression ◽  
Human Monocytic Cell

ABSTRACT Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4+ lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.

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