Viral host-range factor C7 or K1 is essential for modified vaccinia virus Ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit protein kinase R-mediated phosphorylation of eukaryotic translation initiation factor 2alpha.

Vaccinia virus (VACV) infection induces phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), which inhibits cellular and viral protein synthesis. In turn, VACV has evolved the capacity to antagonize this antiviral response by expressing the viral host-range proteins K3 and E3. This study revealed that the host-range genes K1L and C7L also prevent eIF2alpha phosphorylation in modified VACV Ankara (MVA) infection of several human and murine cell lines. Moreover, C7L-deleted MVA (MVA-DeltaC7L) lacked late gene expression, which could be rescued by the function of host-range factor K1 or C7. It was demonstrated that viral gene expression was blocked after viral DNA replication and that it was independent of apoptosis induction. Furthermore, it was found that eIF2alpha phosphorylation in MVA-DeltaC7L-infected cells is mediated by protein kinase R (PKR) as shown in murine embryonic fibroblasts lacking PKR function, and it was shown that this was not due to reduced E3L gene expression. The block of eIF2alpha phosphorylation by C7 could be complemented by K1 in cells infected with MVA-DeltaC7L encoding a reinserted K1L gene (MVA-DeltaC7L-K1L). Importantly, these data illustrated that eIF2alpha phosphorylation by PKR is not responsible for the block of late viral gene expression. This suggests that other mechanisms targeted by C7 and K1 are essential for completing the MVA gene expression cycle and probably also for VACV replication in a diverse set of cell types.

The orthopoxvirus 68-kilodalton ankyrin-like protein is essential for DNA replication and complete gene expression of modified vaccinia virus Ankara in nonpermissive human and murine cells.

Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus (VACV) that is being evaluated as replacement smallpox vaccine and candidate viral vector. MVA lacks many genes associated with virulence and/or regulation of virus tropism. The 68-kDa ankyrin-like protein (68k-ank) is the only ankyrin repeat-containing protein that is encoded by the MVA genome and is highly conserved throughout the Orthopoxvirus genus. We showed previously that 68k-ank is composed of ankyrin repeats and an F-box-like domain and forms an SCF ubiquitin ligase complex together with the cellular proteins Skp1a and Cullin-1. We now report that 68k-ank (MVA open reading frame 186R) is an essential factor for completion of the MVA intracellular life cycle in nonpermissive human and murine cells. Infection of mouse NIH 3T3 and human HaCaT cells with MVA with a deletion of the 68k-ank gene (MVA-Delta68k-ank) was characterized by an extensive reduction of viral intermediate RNA and protein, as well as late transcripts and drastically impaired late protein synthesis. Furthermore, infections with MVA-Delta68k-ank failed to induce the host protein shutoff that is characteristic of VACV infections. Although we demonstrated that proteasome function in general is essential for the completion of the MVA molecular life cycle, we found that a mutant 68k-ank protein with a deletion of the F-box-like domain was able to fully complement the deficiency of MVA-Delta68k-ank to express all classes of viral genes. Thus, our data demonstrate that the 68k-ank protein contains another critical domain that may function independently of SCF ubiquitin ligase complex formation, suggesting multiple activities of this interesting regulatory protein.

Marker gene swapping facilitates recombinant Modified Vaccinia Virus Ankara production by host-range selection.

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental and human vaccine vector for its lack of replication in mammalian cells and high expression of heterologous genes. Recombinant MVA technology can be improved greatly by combining transient host-range selection (based on the restoration in MVA of the deleted vaccinia gene K1L) with the differential expression of fluorescent proteins. Recombinant virus results from swapping a red protein gene (in the acceptor virus) with a cassette of the transfer plasmid comprising the transgene and the green marker K1Lgfp (a chimeric gene comprising K1L and EGFP). Recombinant selection is performed in the selective host RK13. Finally, in the non-selective host BHK-21, a single crossover between identical flanking regions excises the marker gene. The three types of viruses involved (red parental, green intermediate and colourless final recombinant) are visualized differentially by fluorescence microscopy or fluoro-imaging of terminal dilution microcultures, leading to a straightforward and efficient purification protocol. This method (Red-to-Green gene swapping) reduces greatly the time needed to obtain marker-free recombinant MVA and increases the reliability of the construction process.

In vivo attenuation of recombinant murine gammaherpesvirus 68 (MHV-68) is due to the expression and immunogenicity but not to the insertion of foreign sequences.

Recombinant herpesviruses are increasingly utilized to study herpesvirus biology. For recombinant viruses carrying insertions of foreign sequences, attenuated phenotypes in vivo have been frequently observed. In most cases, the underlying mechanisms were not clear or have not been investigated. In this study, we used a recombinant murine gammaherpesvirus 68 (MHV-68), carrying a cassette for the expression of the non-structural protein NS3 of Hepatitis C virus (MHV-68-NS3), to systematically address the question whether the insertion of a defined foreign sequence (NS3) interferes with the biological properties of the recombinant virus in vivo, and to analyze the underlying mechanism. We show that while MHV-68-NS3 is attenuated in vivo, recombinant MHV-68 carrying identical genomic inserts but unable to express the NS3 protein, are not attenuated. Moreover, we provide evidence that the attenuated phenotype of MHV-68-NS3 is caused by the immune response. Our findings are important for the in vivo use of recombinant MHV-68 carrying insertions of marker genes, reporter genes or genes of model antigens. They are also relevant for the potential application of MHV-68 as gene delivery vector.

A prime-boost vaccination protocol optimizes immune responses against the nucleocapsid protein of the SARS coronavirus.

Severe acute respiratory syndrome (SARS) is a serious infectious disease caused by the SARS coronavirus. We assessed the potential of prime-boost vaccination protocols based on the nucleocapsid (NC) protein co-administered with a derivative of the mucosal adjuvant MALP-2 or expressed by modified Vaccinia virus Ankara (MVA-NC) to stimulate humoral and cellular immune responses at systemic and mucosal levels. The obtained results demonstrated that strong immune responses can be elicited both at systemic and mucosal levels following a heterologous prime-boost vaccination protocol consisting in priming with NC protein add-mixed with MALP-2 by intranasal route and boosting with MVA-NC by intramuscular route.

Vaccination with a Modified Vaccinia Virus Ankara-based vaccine protects mice from allergic sensitization.

BACKGROUND: Currently, no treatment is available for food allergy and strict avoidance of the allergenic food remains the only way to manage the allergy. New strategies leading to a safe and efficacious food allergy treatment are required. Modified vaccinia virus Ankara (MVA), which allows high levels of expression of recombinant protein in vivo and gives rise to a Th1-biased specific immune response, was used as a prophylactic vaccine in a murine model of ovalbumin (OVA) allergy. METHODS: An MVA-OVA vector vaccine was prepared. Female BALB/c mice were vaccinated twice with a MVA-OVA vector vaccine, followed by sensitization with OVA plus alum. OVA-specific immunoglobulin E(IgE) activity was measured by mediator release from rat basophilic leukaemia cells, whereas specific IgG subclass titers were determined by enzyme-linked immunosorbent assay. RESULTS: Expression of immunological active OVA in mammalian cells was demonstrated. OVA-specific IgE levels in sera from MVA-OVA-vaccinated mice were reduced and appeared delayed. The vaccine-mediated immune modulation was dose-dependent; the highest vaccine dose protected 50% of the animals from allergic sensitization. Upon sensitization, similar OVA-specific IgG1 titers were found in all mice, but the OVA-specific IgG2a antibody levels were strongly increased in MVA-OVA-vaccinated mice, signifying a Th1-biased and, non-allergic immune response. CONCLUSIONS: Prophylactic vaccination with MVA-OVA delays and in part even prevents the onset of a successful allergen-specific sensitization. Recombinant MVA, which fulfills the requirements for clinical application, is a promising candidate vector for the development of novel approaches to allergen-specific prophylactic vaccination and specific immunotherapy.

The human spleen is a major reservoir for long-lived vaccinia virus-specific memory B cells.

The fact that you can vaccinate a child at 5 years of age and find lymphoid B cells and antibodies specific for this vaccination 70 years later remains an immunologic enigma. It has never been determined how these long-lived memory B cells are maintained and whether they are protected by storage in a special niche. We report that, whereas blood and spleen compartments present similar frequencies of IgG(+) cells, antismallpox memory B cells are specifically enriched in the spleen where they account for 0.24% of all IgG(+) cells (ie, 10-20 million cells) more than 30 years after vaccination. They represent, in contrast, only 0.07% of circulating IgG(+) B cells in blood (ie, 50-100,000 cells). An analysis of patients either splenectomized or rituximab-treated confirmed that the spleen is a major reservoir for long-lived memory B cells. No significant correlation was observed between the abundance of these cells in blood and serum titers of antivaccinia virus antibodies in this study, including in the contrasted cases of B cell-depleting treatments. Altogether, these data provide evidence that in humans, the two arms of B-cell memory--long-lived memory B cells and plasma cells--have specific anatomic distributions--spleen and bone marrow--and homeostatic regulation.

Protective vaccination with hepatitis C virus NS3 but not core antigen in a novel mouse challenge model.

BACKGROUND: Efficient vaccines against hepatitis C virus (HCV) infection are urgently needed. Vaccine development has been hampered by the lack of suitable small animal models to reliably test the protective capacity of immmunization. METHODS: We used recombinant murine gammaherpesvirus 68 (MHV-68) as a novel challenge virus in mice and tested the efficacy of heterologous candidate human vaccines based on modified vaccinia virus Ankara or adenovirus, both delivering HCV non-structural NS3 or core proteins. RESULTS: Recombinant MHV-68 expressing NS3 (MHV-68-NS3) or core (MHV-68-core) were constructed and characterized in vitro and in vivo. Mice immunized with NS3-specific vector vaccines and challenged with MHV-68-NS3 were infected but showed significantly reduced viral loads in the acute and latent phase of infection. NS3-specific CD8+ T cells were amplified in immunized mice after challenge with MHV-68-NS3. By contrast, we did neither detect a reduction of viral load nor an induction of core-specific CD8+ T cells after core-specific immunization. CONCLUSIONS: Our data suggest that the challenge system using recombinant MHV-68 is a highly suitable model to test the immunogenicity and protective capacity of HCV candidate vaccine antigens. Using this system, we demonstrated the usefulness of NS3-specific immunization. By contrast, our analysis rather discarded core as a vaccine antigen.

Evaluation of modified vaccinia virus Ankara as an alternative vaccine against smallpox in chronically HIV type 1-infected individuals undergoing HAART.

The fear of malevolent use of variola virus by terrorists has led to the implementation of a health care worker vaccination program and to the consideration of vaccination for the general public. However, due to concerns about side effects of the classical smallpox vaccine, especially for immunocompromised individuals, a safer vaccine is urgently needed. We characterized the immunogenicity of modified vaccinia virus Ankara (MVA), one of the more promising alternative smallpox vaccines, in a cohort of 10 chronically HIV-1-infected individuals undergoing highly active antiretroviral therapy (HAART). Nine subjects received smallpox vaccination as children while one subject was never vaccinated against smallpox. All the subjects had CD4 counts >400 cells/mm(3) and 8 out of 10 had undetectable viral loads. MVA was able to elicit humoral and cellular immune responses in the majority of individuals. Vaccinia-specific antibodies were mainly of the IgG class while T cells specific to vaccinia were predominantly CD8(+). The immune responses were maintained over 1 year. Similar vaccinia specific humoral immune responses were observed when our cohort of HIV-1-infected individuals was compared to smallpox-vaccinated healthy subjects. The observed immune responses suggest that the highly attenuated MVA could be used as a substitute vaccine against smallpox in chronically HIV-1-infected individuals undergoing HAART.

Recombinant murine gammaherpesvirus 68 (MHV-68) as challenge virus to test efficacy of vaccination against chronic virus infections in the mouse model.

Efficient vaccines against AIDS, Hepatitis C and other persistent virus infections are urgently needed. Vaccine development has been especially hampered by the lack of suitable small animal models to reliably test the protective capacity of candidate vaccines against such chronic viral infections. A natural mouse pathogen such as MHV-68 that persists lifelong after infection, appears to be a particularly promising candidate for a more relevant model system. Here, we investigated infections with recombinant MHV-68 as novel mouse challenge model to test the efficacy of heterologous vaccines based on recombinant modified vaccinia virus Ankara (MVA). To apply ovalbumin (OVA) as a model antigen, we constructed the recombinant virus MHV-68-OVA by BAC technology and characterized genetic stability and replicative capacity of the virus in vitro and in vivo. We demonstrated the ability of MHV-68-OVA to produce ovalbumin upon tissue culture infection. Moreover, the use of MHV-68-OVA-infected target cells allowed for efficient ex vivo amplification of OVA-specific, MHC class I-restricted CD8 T cells derived from MVA-OVA-vaccinated C57BL/6 mice. Finally, we immunized C57BL/6 mice with MVA-OVA and challenged the animals with MHV-68-OVA testing different time points and routes of infection. Vaccinated mice were infected with MHV-68-OVA but showed reduced viral loads in the acute and latent phase of challenge infection. These data strongly suggest the usefulness of the MHV-68 challenge model for further evaluation of recombinant vaccines against persisting virus infections.

Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity.

UNLABELLED: Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P=0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 [corrected] and PD-1 levels in liver correlated with clearance or chronicity. CONCLUSION: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.

Short-term, but not post-exposure, protection against lethal orthopoxvirus challenge after immunization with modified vaccinia virus Ankara.

Safety-tested vaccinia virus (VACV) MVA serves as a candidate third-generation vaccine against smallpox. Here, MVA immunization of mice shortly before or after lethal respiratory challenge with VACV Western Reserve was investigated. Whilst post-exposure treatment failed to protect animals, immunizations on day 2 prior to challenge were fully protective. On the day of challenge, MVA inoculation may prevent death, but not onset of severe respiratory disease. After intranasal MVA application, massive influx of leukocytes (such as neutrophils, macrophages, natural killer cells and T cells) was found in the lungs of the animals, indicating the contribution of innate responses to protection. Correspondingly, in RAG-1-/- mice, MVA inoculation delayed onset of disease significantly, but did not prevent fatal infection. Thus, short-term protection required a tight interplay of both innate and adaptive antiviral immunity. These data suggest that, in addition to conventional vaccination, MVA may serve for potent emergency prophylaxis against orthopoxvirus infection.

Inactivation of the viral interleukin 1beta receptor improves CD8+ T-cell memory responses elicited upon immunization with modified vaccinia virus Ankara.

Interleukin 1 (IL1) is an important regulator of inflammatory responses and contributes to host immune defence against infection. Vaccinia virus encodes a viral soluble IL1beta receptor (IL1betaR), which modulates the acute-phase host response to infection and might influence the induction of immune responses against virus-associated antigens. Here, modified vaccinia virus Ankara (MVA) mutants defective in IL1betaR production were produced by insertion of selectable marker gene sequences that precisely deleted the IL1betaR coding sequences from the MVA genome (MVA-DeltaIL1betaR). Analysis of MVA mutants indicated that deletion of the IL1betaR gene did not abrogate the formation of MVA progeny upon tissue culture propagation. After high-dose intranasal infection with MVA-DeltaIL1betaR, mice showed no signs of fever or other illness, suggesting that the avirulent phenotype remained preserved for MVA-DeltaIL1betaR. Following vaccination of mice, MVA-DeltaIL1betaR or non-mutated MVA induced similar acute-phase immune responses. Importantly, when monitored at the memory phase, significantly higher vaccinia virus-specific total CD8(+) and HLA-A*0201-binding peptide epitope-specific T-cell responses were found after vaccination of HLA-A*0201-transgenic and non-transgenic mice with MVA-DeltaIL1betaR. Moreover, 4-6 months after vaccination, MVA-DeltaIL1betaR provided higher levels of protection against lethal respiratory challenge infection with virulent vaccinia virus strain Western Reserve compared with wild-type MVA. These data suggest that deletion of the viral IL1betaR gene may be considered a relevant approach to amplify the virus-specific CD8+ memory T-cell response and duration of protective immunity obtained after MVA vaccination.

Protective CD8 T cell immunity triggered by CpG-protein conjugates competes with the efficacy of live vaccines.

In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 mug of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.

Role of viral factor E3L in modified vaccinia virus ankara infection of human HeLa Cells: regulation of the virus life cycle and identification of differentially expressed host genes.

Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain being developed as a vaccine for delivery of viral and recombinant antigens. The MVA genome lacks functional copies of numerous genes interfering with host response to infection. The interferon resistance gene E3L encodes one important viral immune defense factor still made by MVA. Here we demonstrate an essential role of E3L to allow for completion of the MVA molecular life cycle upon infection of human HeLa cells. A deletion mutant virus, MVA-DeltaE3L, was found defective in late protein synthesis, viral late transcription, and viral DNA replication in infected HeLa cells. Moreover, we detected viral early and continuing intermediate transcription associated with degradation of rRNA, indicating rapid activation of 2'-5'-oligoadenylate synthetase/RNase L in the absence of E3L. Further molecular monitoring of E3L function by microarray analysis of host cell transcription in MVA- or MVA-DeltaE3L-infected HeLa cells revealed an overall significant down regulation of more than 50% of cellular transcripts expressed under mock conditions already at 5 h after infection, with a more prominent shutoff following MVA-DeltaE3L infection. Interestingly, a cluster of genes up regulated exclusively in MVA-DeltaE3L-infected cells could be identified, including transcripts for interleukin 6, growth arrest and DNA damage-inducible protein beta, and dual-specificity protein phosphatases. Our data indicate that lack of E3L inhibits MVA antigen production in human HeLa cells at the level of viral late gene expression and suggest that E3L can prevent activation of additional host factors possibly affecting the MVA molecular life cycle.

Modified vaccinia virus Ankara as antigen delivery system: how can we best use its potential?

Safety-tested modified vaccinia virus Ankara (MVA) has been established as a potent vector system for the development of candidate recombinant vaccines. The versatility of the vector system was recently demonstrated by the rapid production of experimental MVA vaccines for immunization against severe acute respiratory syndrome associated coronavirus. Promising results were also obtained in the delivery of Epstein-Barr virus or human cytomegalovirus antigens and from the clinical testing of MVA vectors for vaccination against immunodeficiency virus, papilloma virus, Plasmodium falciparum or melanoma. Moreover, MVA is considered to be a prime candidate vaccine for safer protection against orthopoxvirus infections. Thus, vector development to challenge dilemmas in vaccinology or immunization against poxvirus bio-threat seems possible, yet the right choice should be made for a most beneficial use.

Construction and isolation of recombinant MVA.

Modified vaccinia virus Ankara (MVA) is a valuable tool for the expression of recombinant genes used for such purposes as the study of protein functions or characterization of cellular and humoral immune responses. A major advantage of MVA is its clear safety record, and it can be handled under biosafety level 1 conditions. Despite its replication deficiency in human and most mammalian cells, MVA provides high-level gene expression and has proven to be immunogenic when delivering heterologous antigens in animals and humans. This chapter provides state-of-the-art protocols for generation, plaque isolation, molecular characterization, as well as amplification and purification of MVA vector viruses to obtain recombinant viruses for further evaluation.

Neutralization assay using a modified vaccinia virus Ankara vector expressing the green fluorescent protein is a high-throughput method to monitor the humoral immune response against vaccinia virus.

Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.

Vaccinia vectors as candidate vaccines: the development of modified vaccinia virus Ankara for antigen delivery.

Vaccinia viruses engineered to express foreign genes are powerful vectors for production of recombinant proteins. Originating from highly efficacious vaccines securing world-wide eradication of smallpox, the most appealing use of vaccinia vectors is to serve as vaccine delivery system for heterologous antigens. Concerns about the safety of vaccinia virus have been addressed by the development of vectors based on attenuated viruses. One of them, modified vaccinia virus Ankara (MVA) can be considered as current vaccinia virus strain of choice for clinical investigation. Historical development and use of MVA as vaccine against smallpox allowed to establish an extraordinary safety profile. MVA can be used under conditions of biosafety level 1 because of its avirulence and its deficiency to productively grow in human cells. In recent years significant progress has been made with regard to the development of MVA vector technologies. Compared to replication competent vaccinia viruses, MVA provides similar levels of recombinant gene expression even in nonpermissive cells. In animal models, MVA vaccines have been found immunogenic and protective against various infectious agents including immunodeficiency viruses, influenza, parainfluenza, measles virus, flaviviruses, or plasmodium parasites. By now first data from clinical trials are becoming available. In this article we briefly review history of MVA and state-of-the art technologies with regard to generation of recombinant MVA vaccines, and describe the progress to develop MVA vector vaccines against important infectious diseases.